Revision as of 22:32, 17 September 2007 (view source) Rvita (Talk | contribs) (→‎3D Structural Data) ← Older edit		Latest revision as of 21:22, 13 November 2007 (view source) Rvita (Talk | contribs) (→‎Experimental Data)
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	* Data not shown is not curated.		* Data not shown is not curated.
	* Personal communication is not sufficient for curation.		* Personal communication is not sufficient for curation.
		+	*When encountering data from previous publications which are included in figures or tables, the curator should determine if the previous record was curated and if it was not, the PMID should be sent to the Document Specialist to be included in the database.
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	===Scope and Exclusions===		===Scope and Exclusions===

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Latest revision as of 21:22, 13 November 2007

Use of this Manual

The following is a collection of notes from curation meetings with the Epitope Council and is a suggested guideline for capturing immunological data into I.E.D.B.’s Internal Curation System (IPS). The following format will be used in this manual to refer to aspects of IPS data entry:

• The database is structured into categories with headings that contain several fields. Whenever referenced in this manual, [Field Categories] will be in brackets. If a Field Category is mentioned without specifying an accompanying Field, then the guideline applies to all fields within that category.
• Fields in ICS will be underlined.
• "Free Text" and Finder functions will be in quotations.
• <Drop-down> selections from List Fields will be in angle brackets.

Important Note: Section headings highlighted in yellow reflect modifications from the previous version of the curation manual.

Purpose

The purpose of this manual is to ensure consistency and accuracy of literature based curation. All IEDB curators must:

1. Read the manual

2. Read the manual

3. Refer to the manual

4. Follow the manual

5. When experimental scenarios are encountered in the literature that do not conform to the guidelines found in the manual, these issues are to be discussed in the curation meeting to foster development of new guidelines or to adapt our current rules in order to accurately capture epitope related data.

Inclusion/Exclusion Criteria

All articles and epitopes must meet inclusion criteria in order to be included in the database.

Relevant Experimental Data

Experimental Data

The reference must report original and experimental epitope-related data.

• Computer derived predictions without functional experimental data will not be included in the database.
• Sequence analysis of defined epitopes will not be included in the database unless novel information is provided (e.g., identification of anchor residues).
• Reviews and meta analysis will not be included in the database.
• Data not shown is not curated.
• Personal communication is not sufficient for curation.
• When encountering data from previous publications which are included in figures or tables, the curator should determine if the previous record was curated and if it was not, the PMID should be sent to the Document Specialist to be included in the database.

Scope and Exclusions

The experimental data must fall within the scope of the database.

Relevant data includes:

• MHC binding data
• Epitope elution from MHC (Naturally processed MHC ligands)
• T cell responses to an epitope (including NK T cells)
• B cell/antibody responses to an epitope

Certain categories of experimental data will be specifically excluded from the database.

Exclusion: NK Epitopes

Epitopes that are recognized by Natural Killer cells (non-T cell) will be excluded from the database. Please note: NK T cell epitopes/ligands will be included (as noted in section (#Scope and Exclusions).

Exclusion: Non-Immunological Interpretation of "Epitope"

Experimental data describing "epitopes" in non-immunological contexts will not be included in the database. For example, the structures that are in contact in protein-protein interactions are sometimes referred to as an epitope. This interpretation of "epitope" will not be included in the database.

Exclusion: Epitope Tags

References discussing epitope tags utilized as a technical tool for immunoprecipitation, purification, and similar experiments will be excluded from the database.

Exclusion: Superantigen

References discussing superantigens in the context of peptide-MHC-super antigen complexes will be excluded from the database. However, B cell/antibody responses to superantigens (especially Staphylococcus enterotoxin B (SEB), a NIAID Category B priority pathogen) will be curated.

Important Note: In the event superantigen is used as part of an assay to stabilize the interaction of an epitope with MHC, the epitope studied is curatable and should be captured.

Exclusion: TCR Antagonism

Data related to TCR antagonism will not be curated. Author stated TCR antagonism will not be curated, however, TCR competition not specifically labeled as TCR antagonism by the authors will be curated. TCR antagonism used in an MHC binding inhibition assay will be captured as an MHC binding assay.

Exclusion: Antigen Processing

Data concerning the processing of antigens generated in order to study the effects of variables on processing rather than on the study of an epitope (i.e. the epitope is irrelevant) are not to be included in the database unless the identification of a novel epitope is demonstrated.

Exclusion: Adoptive Transfer

Assays involving adoptive transfer will not be curated at this time.

Epitopes Relevant to IEDB

Length/Mass Restrictions

Table 1. Length/Mass Restrictions

Epitope Class	Uncuratable	Epitopic Region (# residues)	Epitope (# residues)
B-cell	> 5 kDa or 50 amino acids	12 - 50	1 ≤ x ≤ 11
Class I	> 5 kDa or 50 amino acids	12 - 50	7 ≤ x ≤ 11
Class II	> 5 kDa or 50 amino acids	16 - 50	7 ≤ x ≤ 15

The database will only include epitopes of less than 50 residues in either a linear or conformational sequence. If the epitope is non-peptidergic, the mass restriction is to be less than 5000 Daltons to be included in the database.

Important Exception: Epitopes greater than 50 residues will be curated for certain pathogens including Botulinum toxin and anthrax epitopes.

A region or fragment of >50aa from B. anthracis and C. botulinum will be curated as an epitopic region in the following cases:

Well-Characterized Epitopes

To broaden the spectrum of information in the database, we currently exclude the repeated curation of epitopes once 10 key references have been included in the database. The original articles describing the epitope, MHC restriction data, antibody responses, and articles containing novel information regarding the epitope will be included in the approximately 10 references. A compiled list of "well-characterized" epitopes is listed in Table 2 and can also be found in the Curation Network folder (\\Curation\CurationNotes\blacklisted.xls).

Table 2. Well-characterized epitopes

#	Common Name	Sequence	Positions	Source Species	Source Protein Name	Restriction Allele
1	TT Universal Helper epitope	QYIKANSKFIGITE	830-843	Clostridium tetani	Tetanus toxin	DRB1*1302
2	OVA 257-264	SIINFEKL	257-264	Gallus gallus (Chicken)	Ovalbumin	Kb
3	Ova 323-339	ISQAVHAAHAEINEAGR	323-339	Gallus gallus (Chicken)	Ovalbumin	H2-Ag7, RT1.B1
4	HEL 46-61	NTDGSTDYGILQINSR	46-61	Gallus gallus (Chicken)	Hen Egg white Lysozyme	H-2 IAk
5	HBV core 18-27	FLPSDFFPSV	18-27	Hepatitis B	Core Protein	A2
6	SL9	SLYNTVATL	77-85	HIV	Gag-p17	A*0201
7	TAX 11-19	LLFGYPVYV	11-19	HTLV	Transcriptional activator (tax)	A*0201
8	SYFPEITHI	SYFPEITHI	367-375	Human	Tyrosine kinase JAK1	Kd
9	MART-1(27-35)	AAGIGILTV	27-35	Human	MART-1 (Tumor antigen)	A*0201
10	NY-ESO-1 epitope	SLLMWITQC	157-165	Human	Cancer/testis antigen NY-ESO-1	A*0201
11	Tyrosinase 370D	YMDGTMSQV	368-376	Human	Tyrosinase (Tumor antigen)	A*0201
12	gp100 210M	IMDQVPFSV	209-217	Human	Glycoprotein (melanocyte lineage-specific antigen)	A2
13	HER-2/neu 689-697	RLLQETELV	689-697	Human	HER-2/neu (Tumor antigen)	A*0201
14	HER-2/neu 369-377	KIFGSLAFL	369-377	Human	HER-2/neu (Tumor antigen)	A*0201
15	PLP 136-151	HSLGKWLGHPDKF	136-151	Human	Myelin proteolipid protein	H-2 IAs
16	MBP Ac1-11	Ac-ASQKRPSQRSK	1-11	Human	Myelin Basic protein	H-2 IAu
17	CMV pp65	NLVPMVATV	495-503	Human cytomegalovirus	Phosphoprotein 65 (pp65)	A*0201
18	M1	GILGFVFTL	58-66	Influenza	Matrix Protein	A*0201
19	Flu HA 307-319	PKYVKQNTLKLAT	307-319	Influenza	Haemagglutinin	DRB1*0101, DRB1*0401 (DR4Dw4), DRB1*0701, DRB1*1101, DRB5*0101(DR2a)
20	PA 224-233	SSLENFRAYV	224-233	Influenza	Acid Polymerase	H-2 Db
21	NP 366-374	ASNENMETM	366-374	Influenza	Nucleoprotein	H-2 Db
22	NP 147-155	TYQRTRALV	147-155	Influenza	Influenza Nucleoprotein	H-2 Kd
23	HA 110-120	SFERFEIFPKE	110-120	Influenza	Haemagglutinin	H-2 IEd
24	LCMV gp33	KAVYNFATC	33-41	LCMV	Glycoprotein	Kb, Db
25	LCMV gp 276	SGVENPGGYCL	276-286	LCMV	Glycoprotein	Db
26	LCMV np 396	FQPQNGQFI	396-404	LCMV	Nucleoprotein	Db
27	LCMV np 118-126	RPQASGYM	118-126	LCMV	Nucleoprotein	H-2 Ld
28	LLO 91-99	GYKDGNEYI	91-99	Listeria monocytogenes	Listeriolysin O	H-2 Kd
29	LLO 215-226	SQLIAKFGTAFK	215-226	Listeria monocytogenes	Listeriolysin O	H-2 IEk
30	LLO 190-201	NEKYAQAYPNVS	190-201	Listeria monocytogenes	Listeriolysin O	H-2 IAb
31	P60 449-457	IYVGNGQMI	449-457	Listeria monocytogenes	p60	H-2 Kd
32	P60 217-225	KYGVSVQDI	217-225	Listeria monocytogenes	p60	H-2 Kd
33	f-MIGWII	MIGWII	1-6	Listeria monocytogenes	LemA	H2-M3
34	NANP	NANP		Plasmodium falciparum (malaria)	Circumsporozoite protein
35	MCC 88-103	SYIPSAEKI	252-260	Plasmodium berghei	Circumsporozoite protein	H-2 Kd
36	MCC 88-103	ANERADLIAYLKQATK	88-103	Macrobrachium malcolmsonii (moth)	Cytochrome c	H-2 IEk
37	Hsp 234-252	LREAAEKAKIELSSSQSTS	234-252	Mycobacterium tuberculosis	Heat shock protein (HSP) 70	RT1.B
38	PCC 88-104	KAERADLIAYLKQATAK	88-104	Pigeon	Cytochrome c	H-2 IEk

The above list plus any well characterized epitopes from additional sources encountered as the range of curated subjects is expanded are to be curated following the exceptions noted below. We avoid curating papers that focus on exploring strategies to enhance well characterized epitope's immunogenicity. These papers use well-characterized peptides to evaluate variables such as alternative immunogen constructs (MAPS/vectors) or adjuvants.

Important Note: When a well-characterized epitope is presented in the context of an epitopic region or domain, the longer peptide will be considered a well-characterized epitope.

Important Exceptions:

• When well-characterized epitopes are studied in a novel host in terms of the MHC, TCR, antibody, or otherwise, the reference will be included in the database.
• When well-characterized epitopes are used as controls they will not be curated. However, when the well-characterized epitope is used as a reference for a novel epitope from the same source protein, the well-characterized epitope will also be captured.
• When a reference describes a well-characterized epitope in addition to other curatable epitopes, all of the epitopes from the reference will be captured.
• At the curators/EC discretion, papers describing new/additional original data relating to these epitopes can and will be curated. For example, if the paper make a novel discovery about the epitope.

HIV/SIV

References describing only epitopes derived from HIV/SIV will not be included in the database. However, when a manuscript describes epitopes from other relevant sources as well as HIV/SIV, all of the epitopes in the manuscript will be captured.

Imported Data

When curating data previously imported from other databases, the curator should apply all curation rules and recurate any data as needed in order to comply with IEDB rules. This includes entering new epitopes, deleting epitopes not conforming to our criteria, and adding any new contexts as needed. References which originate from an import should be labeled as such on the reference page Each imported epitope should also be labeled as originating from a data import. For example, a phrase such as "Data originally imported from the HLA Ligand Database" should be entered whenever applicable.

Epitopes entered through direct submission will not be recurated, rather the publication regarding the directly submitted data will be curated as a new reference. Knowledge of the directly submitted data can be helpful in gathering epitope sequences and checking epitope structural data.

Curation Prioritization

Epitope curation should be conducted in the following priority order:

A) NIAID Category A, B, and C priority pathogens and toxins:

The complete list of NIAID Category A, B, and C priority pathogens and toxins can be found at the following URL: http://www2.niaid.nih.gov/biodefense/bandc_priority.htm.

NIAID – Category A

Bacillus anthracis (anthrax)

Clostridium botulinum

Yersinia pestis

Variola major (smallpox) and other pox viruses

Francisella tularensis (tularemia)

Viral hemorrhagic fevers

Arenaviruses

LCM, Junin virus, Machupo virus, Guanarito virus

Lassa Fever

Bunyaviruses

Hantaviruses

Rift Valley Fever

Flaviruses

Dengue

Filoviruses

Ebola

Marburg

NIAID – Category B

Burkholderia pseudomallei

Coxiella burnetii (Q fever)

Brucella species (brucellosis)

Burkholderia mallei (glanders)

Ricin toxin (from Ricinus communis)

Epsilon toxin of Clostridium perfringens

Staphylococcus enterotoxin B

Typhus fever (Rickettsia prowazekii)

Food and Waterborne Pathogens

Bacteria

Diarrheagenic E.coli

Pathogenic Vibrios

Shigella species

Salmonella

Listeria monocytogenes

Campylobacter jejuni

Yersinia enterocolitica)

Viruses (Caliciviruses, Hepatitis A)

Protozoa

Cryptosporidium parvum

Cyclospora cayatanensis

Giardia lamblia

Entamoeba histolytica

Toxoplasma

Microsporidia

Additional viral encephalitides

West Nile Virus

LaCrosse

California encephalitis

VEE

EEE

WEE

Japanese Encephalitis Virus

Kyasanur Forest Virus

NIAID – Category C

Tickborne hemorrhagic fever viruses

Crimean-Congo Hemorrhagic fever virus

Tickborne encephalitis viruses

Yellow fever

Multi-drug resistant TB

Influenza

Other Rickettsias

Rabies

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

B) Emerging and Re-emerging pathogens:

Pathogens newly recognized in the past two decades

Acanthamebiasis

Australian bat lyssavirus

Babesia, atypical

Bartonella henselae

Ehrlichiosis

Encephalitozoon cuniculi

Encephalitozoon hellem

Enterocytozoon bieneusi

Helicobacter pylori

Hendra or equine morbilli virus

Hepatitis C

Hepatitis E

Human herpesvirus 8

Human herpesvirus 6

Lyme borreliosis

Microsporidia

Parvovirus B19

Coronaviruses/Severe Acute Respiratory Syndrome (SARS)

Re-emerging Pathogens

Enterovirus 71

Prion diseases

Streptococcus, group A

Staphylococcus aureus

• Coccidioides immitis

C) Transplant rejection antigens and other alloantigens, Allergens, and Self antigens involved in autoimmunity

D) Infectious diseases not listed above under sections (#Curation Prioritization) A) and B)

E) Epitopes associated with cancer

Minimal Data Requirements

The reference must contain information for all required fields for at least one epitope in order to be included in the database. There are five major categories containing twenty-six fields. One set of data from each category must be available. These fields are highlighted in yellow in the data dictionary available in the Curation Network folder: (\\Curation\Docs\DataDictionary\). The required fields are listed in Table 3

Table 3 Required Fields

#			Section Classification	Field Name	Comments
1	a		Reference -Journal Article	PubMed ID	At least one set of fields from Category # 1 (1a, 1b or 1c) has to be filled out.
	b	i	Reference - Submission	Author(s)
		ii	Reference - Submission	Affiliation(s)
	c		Reference - Patents	Paten Publication Number
2	a		Epitope Structure	Linear Sequences	At least one of the three fields from Category # 2 (2a,2b or 2c) has to be filled out.
	b		Epitope Structure	SMILES Structure
	c		Epitope Structure	Conformational Sequence
3	a		Epitope Structure	Epitopic Region / Domain	Mandatory field. This Boolean field indicates whether the epitope that is captured is a minimal epitope or contained within a region / domain.
4	a		Epitope Source	Epitope Source Nature	At least one of the seven fields from Category # 4 (4a, 4b, 4c, 4d, 4e, 4f or 4g) has to be filled out. If the value of natural Antigen, which is a Boolean field, is ’no’, all other Epitope-Source fields are ignored.
	b		Epitope Source	Source Species
	c		Epitope Source	Gene Name
	d		Epitope Source	Protein Name
	e		Epitope Source	GenBank ID
	f		Epitope Source	Swiss Prot ID
	g		Epitope Source	PDB ID
5	a	i	MHC Binding	MHC Allele	At least one set of fields from Category # 5 (5a, 5b, 5c, 5d) has to be filled out. All the fields in a subsection has to be filled out if that subsection is selected. For example, if 5a is chosen, all three fields (5a-i,ii,iii) have to be filled out. The following fields - Assay Type, and Qualitative Measurement, can be entered as "Unknown" if the data is unavailable. It’s anticipated that most the data imports from other existing databases might not have the assay related fields
		ii	MHC Binding	Assay Type
		iii	MHC Binding	Qualitative Measurement
	b	i	MHC Ligand Elution	MHC Allele
		ii	MHC Ligand Elution	Assay Type
		iii	MHC Ligand Elution	Qualitative Measurement
	c	i	T Cell Response - Assay	MHC Allele
		ii	T Cell Response - Assay	Assay Type
		iii	T Cell Response - Assay	Qualitative Measurement
	d	i	B Cell Response - Assay	Assay Type
		ii	B Cell Response - Assay	Qualitative Measurement

Epitope Structure Availability

In the event the exact epitope structure is not given in the reference, follow these guidelines:

I) Contact the corresponding author(s) using the template contact letter provided in the Curation Folder based upon information given in the manuscript. In the internal Curation Tracking System (CTS):

A) Fill out F2: Status should be "Waiting for author’s response". Enter author’s e-mail address and date of e-mail in Comments section of F2 form.

B) Once author provides structure(s): Complete the curation of the article, including "Provided by author" in the Data Location Field in Epitope Structure.

C) Update F2, adding comments to include by whom and when the information was sent.

II) If an e-mail address is not provided in the article, a reference cited by the manuscript regarding the epitope structure may be used for sequence information. The [Epitope Structure] Data Location Field should state where the information was found as cited reference or author communication.

The following format should be used anytime literature is cited in the IEDB: Sette et al. (2007). Nature 48: 1141-7. [PMID:12345678]

When citing epitope location, “Reference cited” should be used in location field and citation information should be placed in comments. Provide the PMID whenever it can be easily obtained.

III) If doubts persist regarding the epitope structure, the data cannot be included in the database. If the author does not respond within two weeks, the reference will be deemed uncuratable. Curation status in Form F2 should be selected as <Uncuratable: Reference Scan> with comments including the reason(s) why the epitope/reference was deemed uncuratable.

Important Note: Cited References The same guidelines are to be applied when researching context information such as the generation of T cell clones or monoclonal antibodies. When researching clone production and mAb generation, only go to references cited by the paper that you are curating. If those references do not provide the needed information, do not continue to search further. Add an immunization comment stating that the details were not provided and clarify this point to the reviewer on the cover sheet, letting the reviewer know that you did look at the references, but still could not find the needed information.

How to Curate

General Considerations

The Curation Notes focus on detailed rules for handling data related to specific topics such as epitope structure, B and T cell responses, and negative data. When approaching a curation, one must take into consideration the main conclusions of the paper as revealed in the title and abstract. Rather than being buried in the comments, the main conclusions should be clear in the database and easily available to the user in a search. Be careful to avoid omission of critical data by blindly following curation rules. Be true to the integrity of the data and always consider the end user’s perspective.

Prevailing Rules

Certain database rules supersede all other criteria. Consider the full scope of the data prior to curation.

• Due to the broad specificity of MHC molecules, all MHC binding data is captured. All MHC data receive separate entries and are not candidates for bulk curation.
• All naturally processed epitope data is captured. All naturally processed data receive a separate entry and are not a candidate for bulk curation.
• Do not capture positive or negative controls
• Do not repeat data. If the database fields are used properly, each type of data should be entered only once, in its proper field.
• Comments are to only be used when needed to clarify the information present in the other fields, not to repeat the information present in the other fields.

Comments

There are comment fields throughout the database in order to give the curator the ability to capture VITAL information that would otherwise not be captured by the available fields of the database. The following guidelines should be used when entering comments.

Comments are not required and should always be as brief as possible. Comments should only contain information specific to the pertinent field. In other words, the Comments on Assay field should provide only details pertinent to the assay, its results, or interpretation.

• Comments should stand alone and be self-explanatory. The database user should not need to obtain the actual reference in order to understand the comments. Do not refer to figures in a manuscript in the comments fields unless the accompanying comment remains self-explanatory even if the data location were to be removed. The preferred format for alluding to other figures or tables is in parentheses or braces at the end of the sentence.
• Be tactful and neutral. Refer to any unavailable or unclear information in an inoffensive manner. The fact that particular information is not available in the manuscript does not require mention unless it would be confusing for the end user otherwise. These fields are not meant to explain curation strategies to reviewers, but to convey critical information to end users.
• Avoid the use of uncommon abbreviations. All users should be able to understand the comments. Acceptable abbreviations used in the database will conform to a standard abbreviation list compiled from five journals (J Immunol, PNAS, J Virol, Eur J Immunol and Cancer Immunity). A compiled list of standard abbreviations can be found in Appendix (Section (#Standard Abbreviations) )
• Comments should be written in proper American English to ensure consistency and allow for standardized querying. Check spelling and grammar and use complete sentences (noun and verb). Remember to use articles such as of and the. Capitalize the first letter of the first word in a sentence and only capitalize appropriate words in the text (T cell, M. tuberculosis). Sloppy writing reduces the credibility of the database. Do not use contractions.
• Do not describe routine details of the assay. These fields are not meant to describe the experimental protocol, but rather to clarify what is present in the other fields and are to be used only when needed.
• Do not repeat the information already captured in other fields.
• Avoid interpretation of the manuscript. It is preferable to paraphrase author’s interpretations or conclusions from article text.

Specific Fields

Epitope Structure

This section captures the molecular structure of an epitope, which is defined as the structure interacting with receptors of the immune system (T cell, B cell/ antibody, MHC).

Analogs

Analogs are synthetic constructs of peptide sequences or chemical compounds that share some structural features in common with another sequence or compound. They are often used to determine the role of specific amino acids in the binding or immunogenicity of an epitope. The source of an analog is always artificial.

Analogs are not captured as separate epitope entries, but are referenced in Comments on Assay fields or entered as a context of the wild type sequence UNLESS they are used in MHC binding contexts or are used in assays that do not involve the wild type sequence present in the form of epitope, source antigen, or source species used as either the antigen or the immunogen. See sections (#Key Residues) or (#Linear Epitopes) for further instructions on strategies for curation of many commonly encountered analogs.

All analog sequences accompanied by MHC binding data or sequences that are designed to improve upon the immunogenicity of an epitope must be entered as separate epitope entries. Additionally, all contexts that do not use the natural epitope as either the immunogen or antigen, but instead use only the analog sequence will also require the analog be entered as a separate epitope entry. An example of such an assay would be the use of a CTL line that was raised to the analog lysing APC presenting the analog.

Be sure to comment in the Epitope Comments field what wild type source the analog was derived from in order to retain some source information for the artifical epitope.

Modified Amino Acids

The Post-Translational Modification Type field is used to describe modified amino acids (naturally-occurring, post-translational modifications, or chemical/synthetic modifications). Here is an example for an N-formylated peptide:

Do not use designate the formyl-methionine residue as "fM" as shown in Figure 2 since it conflicts with our use of lower case symbols to refer to D-amino acids. Instead, enter the N-formylated peptide sequence information into ICS as illustrated below (Table 4 ):

Table 4. Example for Entering Modified Amino Acids

Field	Text
Linear Sequence	MIGWII
Modification Type	<Formylation |FORM>
Modified Sequence/Residue	M1

In the Modified Sequence field, use the single letter amino acid code followed by the position of the residue in the epitope at it is captured in the Linear Sequence field. At present ICS does not accommodate the entry of multiple modification types. When a reference describes multiple modification types, consult with a senior curator or Epitope Council member to complete the curation. IEDB conforms to SWISSPROT when capturing post-translational modification types (http://www.expasy.org/tools/findmod/findmod_masses.html). For other modification types, it is added to the drop-down as specified in the reference after consultation with a senior curator.

Important Note: In the event a reference describes the use of an unmodified epitope and additionally that same epitope in a modified form (glycosylated, ubiquitinated, etc), the modified epitopes are to be curated following the rules used to curate analogs (#Analogs). They will not be captured as a separate epitope entry unless the criteria mentioned in (#Analogs) is met.

Important Note: All modifications are to be entered via the Modification Type field and not in the SMILES Structure field.

Important Note: Do not guess the correct modification to use. If the exact term that the authors used is not present, refer to a senior curator regarding synonyms for modifications or the need to add new terms to the drop down list.

Mimotopes

Mimotopes are functional mimics of natural molecular structures which bear little or no sequence homology to their biological counterparts. Mimotopes should be captured as separate epitope records. Follow the guidelines below to capture mimotopes. If a biological homolog is identified from the mimotope structure and is tested, the naturally occurring sequence should also be captured as a separate epitope context.

• Enter the mimotope sequence or structure in the appropriate Sequence fields (Linear Sequence, Conformational Sequence or SMILES structure).
• Specify "Yes" in Author Identified Mimotopes field.
• In the [Epitope Structure] Comments field record the name or give brief information regarding the naturally occurring structure that the mimotope mimics.
• If the mimotope has a natural source antigen, specify those details using the [Source Antigen] fields.
• If the mimotope does not have a natural source, select the [Source Antigen] as either No Natural Source or Phage Display from the source finder. <Phage display library> has the accession number of SRC-1627.

Amino Acid Configuration

The amino acids recorded in Linear Sequence and Conformation Sequence fields are assumed to be in the L-amino acid configuration by default. If a D-amino acid configuration is reported, record the corresponding D-amino acids in "lower" case (SIINFeKL instead of SIINFEKL for a D-glutamate residue) and mention in the Epitope Comments field that the amino acids in the lower case are in the D-amino acid configuration.

Key Residues

When critical residues for the recognition of a linear epitope by T cell or B cell receptors, antibody, or MHC are described in a reference, this information is context dependent. Use the Assay Comments field to describe the key residues. The experiments identifying key residues of linear epitopes are not captured as separate contexts.

Important Exception: When conformational epitopes are defined in a reference by only key residues, the key residues should be entered in the Conformational

Sequence field. Thus, in assays identifying a single amino acid as a key residue, that amino acid will be entered in the Conformational Sequence field unless there is evidence that it is part of a linear epitope.

We assume that a mAb recognizing a conformational epitope defines the epitope, unless otherwise specifically stated by the authors.

Linear Epitopes

When alanine-scanning mutagenesis or other residue substitutions are used to determine key residues within a linear epitope sequence, the key residues should be captured in the Comments on Assay field.

The sequences utilized to deduce key residues are not captured in separate contexts. Rather, in the Comments on Assay field, capture in one or two short and concise statements how the residues were determined to be key. Use standard amino acid notation to denote key residues (Amino acid one-letter code and its residue number e.g. L107). The principal prerequisite for capturing epitopic determinant data is the demonstration of antibody or TCR binding to the native epitope sequence with appropriate controls. All MHC binding data and all naturally processed data are always curated, even when used to deduce key residues.

Figure 3 (pasted from reference with PubMed ID: 15213134) shows an example of alanine substitution data that should not be curated as separate contexts, but may be summarized as a comment. The Epitope Comments field should state "Alanine substitutions for residues F295, T300, Y301, and Y302 eliminated T cell activation, identifying these as critical amino acid residues in the epitope".

Conformational Epitopes

When capturing conformational epitopic determinants, Select <Discontinuous> in the Continuous sequence ? field and then enter the residues into the Conformational sequence field utilizing standard amino acid notation. If the positions listed by the author for the involved residues do not match the positions of those same residues in the Swiss-Prot source, enter the Swiss-Prot numbering along with the residues utilizing standard amino acid notation in the Swiss-Prot Positions field.

Important Note: It is important to always check the source cited for discontinuous epitopes to determine if the residues you are entering do indeed occur and at what positions they occur in the source.

The primary data to capture is a demonstration of antibody binding to the native sequence in non-denaturing assays (ELISA, X-Ray Crystallography, NMR).

As with linear epitopic determinants, the peptide sequences of all mutations or variants tested are not captured as individual epitopes. Enter relevant details regarding the variants or mutants in the Comments on Assay field and information regarding the determination of the epitope structure in the [Epitope Structure] Comments field.

Viral Escape Mutations

When entering viral escape studies into the database, the emphasis should be on antibody binding to residues of the wild-type sequence.

Binding of a monoclonal antibody (mAb) or antisera to the wild-type sequence must be demonstrated along with a loss of binding to escape mutants in order for the data to be included in the database. The negative binding data of the mAb or antisera to escape mutants is not curated.

Each mAb should be treated as though it defines a separate epitope, unless explicitly stated otherwise by the authors. If the panel of monoclonal antibodies is used to characterize binding to an antigen, follow the guidelines described in section (#Panel of Monoclonal Antibodies).

Multiple amino acid residues identified by a series of binding experiments may be captured as a single entry in the Conformational Sequence field if identified as belonging to a single epitope by the authors. Individual binding experiments (inhibition, neutralization, ELISA) will be captured as separate contexts of the conformational epitope.

It is not necessary to capture the substituted amino acid residues found in mutants; however, this information can be entered in the Comments on Assay field.

Information regarding the method used to determine the epitope sequence should be summarized in the [Epitope structure] Comments field.

Deduced Epitopes

A deduced epitope is one that is not directly tested as an isolated structure , but rather deduced by the authors by various methods such as the use overlapping peptide scans. Deduced epitopes may be curated, however, those epitopes defined only by computer prediction algorithms in the absence of validating experimental data will not be included in the database. Follow the flow chart depicted in Figure 4 to determine how to curate deduced epitopes.

Additionally, when curating the use of multiple peptides (as antigen) in order to deduce an epitope, only one context should be curated using the optimal or minimal peptide as the antigen with a comment regarding the use of multiple peptides.

Minimal Epitopes vs Optimal Epitopes

The minimal epitope is the shortest length or smallest structure that produces a cellular or humoral response (is immunogenic) or can serve as an antigen (is antigenic). For MHC restricted responses, a minimum of 7aa are required for a sequence to be considered an epitope (see guidelines in section (#Truncation)).

The authors may describe the epitope with the greatest response as the optimal epitope. There are cases where the minimal epitope does not necessarily give the optimal response. This situation is common with Class II and Antibody/B cell epitopes.

In the event the minimal epitope is not the optimal epitope, the optimal epitope will be captured instead of the minimal epitope. For example, in Figure 5 (PubMed ID: 15048720) the optimal responses in panels a and c are produced by the longer peptides (represented by diamond symbols) and thus are not the minimal epitopes. In this instance, the optimal epitopes in panels a and c will be captured instead of the minimal epitopes.

Minimal Epitope vs Fine Specificity

Fine specificity refers to the detailed pattern of reactivity of different T cell clones or monoclonal antibodies when recognizing the same epitope due to changes of the components such as individual amino acids or sugars within an epitope. Typically, multiple T cell clones and monoclonal antibodies will recognize different amino acids as key residues within the same stretch of the protein sequence. Follow the guidelines below when there are fine specificities reported for a group of T cell clone or monoclonal antibodies.

• Class I epitopes: if T cell clones respond optimally to amino acid sequences of different lengths in a truncation analysis, each of these sequences will be entered as separate epitopes, even though they may contain the same core sequence.
• Class II epitopes: if multiple T cell clones respond to an amino acid sequence that satisfies the criteria of an epitope (e.g. 15 amino acids or less), but differ in their individual reactivities within that stretch of amino acids (e.g. clone 1 recognizes aa 1-13 and clone 2 recognizes aa 3-15), then the longer sequence containing all of the residues recognized by all of the clones will be entered as a single epitope (e.g .aa 1-15). This is considered a single epitope with different fine specificities.
• B cell epitopes: Linear Antibody/B cell epitopes will be entered in the database similarly to Class II epitopes as above. For conformational Antibody/B cell epitopes, assume that each mAb defines its own epitope unless otherwise indicated by data or stated by the author.

These guidelines will be overruled when the authors consider the epitopes distinct, in which case they will be entered as individual epitopes.

Epitopic Region/Domain

The Epitopic Region/Domain field indicates whether the epitope is a minimal epitope or is contained within a region or a domain. The choices are Defined Epitope, Epitope Containing Region/Antigenic Site, and Residues Involved In Recognition.

Define the epitope as a defined epitope if the authors state that it is defined, optimal, or minimal. If the authors do not specify whether the epitope is minimal or not, then use the Table in Section (#Length/Mass Restrictions) and also the guidelines developed by the IEDB Epitope Council below to determine the correct designation:

• For linear Antibody/B Cell and Class I epitopes, if the sequence is 11 residues or less in length (with a minimum of 7 aa for class I epitopes, see (#Truncation)), the epitope should be designated as a Defined Epitope. If the sequence is 12 residues or greater, Epitope Containing Region/Antigenic Site should be selected.
• For linear Class II epitopes, select Defined Epitope if the sequence is 15 residues or less in length. Select Epitope Containing Region/Antigenic Site if the epitope sequence is 16 residues or greater in length.
• For nonlinear/discontinuous epitopes and the curation of key residues, select Residues Involved In Recognition, Epitope Containing Region/Antigenic Site or Defined Epitope as applicable.
• For epitopes with both Class I and Class II contexts or both B cell and T cell contexts, define the epitope under the positive context. In the event both B and T cell assays are positive, define the epitope under the T cell guidelines.

Important Note: The size criteria used to determine epitopic region designations apply regardless of the qualitative data present for the epitope. For example, if only negative data is present in the reference for a particular structure, the designation as Defined Epitope will still be used if the size criteria are met. The external database will define structures having no positive contexts as "distinct structures" rather than "distinct epitopes".

Important Note: Residues involved in recognition are not captured when they are involved in the structural features of the recognition. That is, if a mutation results in a significant structural change, this mutation cannot be assumed to be involved in recognition by the antibody or TCR and is not captured as an epitope.

Ambiguous Cases for Designating a Sequence as an Epitope

Typically the assignment of a sequence as an epitope is unambiguous. However there are a number of scenarios in which more than one structure may be assigned as the epitope within the context of a single assay. Accordingly there may be ambiguity in deciding which structure is to be considered the epitope.

• The sequence or molecular structure of an epitope may be shown to be conserved in several different antigens and in different species. The database only allows reference to one source antigen/source species per epitope entry. In order to enter multiple source species, multiple epitope entries are required.
• Cross-reactivity studies may utilize an epitope molecular structure conserved across multiple source antigens or species; however, only reference to one source may be used per epitope entry in ICS.
• Experimental data might reflect the use of an immunogen and an antigen derived from different source antigens or species, creating confusion regarding whether the immunogen or antigen should be designated as the epitope under which the experimental data will be curated.

Use the following guidelines to determine the structure to be entered as the epitope:

Case 1: If both the immunogen and the antigen are naturally existing molecular structures or both are artificial and one of them is an epitopic region while the other is a minimal epitope, the minimal epitope will be the captured epitope and is entered in the [Epitope Structure] category.

Case 2: If the immunogen and the antigen are both naturally existing molecular structures or both are artificial and both of them are minimal epitopes, then both sequences will get captured in separate entries as epitopes. The end result of this is that each epitope will receive an identical copy of the assay context. Duplication of assay contexts is necessary to insure that both epitopes receive equal priority in the database.

Case 3: If either the immunogen or antigen is artificial, that is, it does not exist in nature while the other is a naturally existing molecular structure, the natural structure will be designated as the epitope and entered in the [Epitope Structure] category. The artificial antigen or immunogen will be specified in the context (as assay antigen or immunogen) of the curated natural epitope.

Important Note: When an epitope sequence is present in more than one source and the other potential sources of the epitope are not specifically tested, but rather this information is only commented upon, the epitope is assigned to the source which was tested in the reference. In contrast, if several natural sources containing the epitopic sequence are studied in the reference, the epitope should be curated under separate entries for each source.

Conservancy/Cross-Reactivity

These guidelines determine the process used to curate data relating to conservancy and cross-reactivity.

This section is being reevaluated in light of all other rules regarding cross reactivity, novelty points, etc. New guidelines may soon appear.

Case 1: When an epitope is analyzed for conservancy or cross-reactivity among different natural proteins or pathogens with experimental data presented in the reference for each of the different sources, the different structures should be entered in the database as separate epitopes for each of the different natural proteins or pathogens according to section (#Ambiguous Cases for Designating a Sequence as an Epitope).

Case 2: When sequence or homology analysis of an epitope and/or source protein data is present, the significance of the data should be mentioned in the [Epitope Structure] Comments field.

Case 3: When an artificial peptide is used for conservancy analysis among multiple proteins / source species, only ONE [Epitope Structure]/[Source Antigen] pair is curated with all others recorded in the Comments on Assay field. The significance of the data will be used to determine the chosen epitope-source antigen pair.

Important Note: These cross-reactivity and conservancy rules are applied only to different species and NOT to different strains. Different strains are curated according to the guidelines specified in section (#Decision Scheme for Bulk Curation).

Epitope Source/Source Antigen

The natural source from which the epitope was derived is always entered as the [Source Antigen] for the epitope. For example, if a gene encoding for a peptide in the Hepatitis A Virus envelope protein is inserted into a Vaccinia virus vector, the [Source Antigen] of the epitope is the Hepatitis A Virus envelope protein rather than the Vaccinia virus vector.

The protein ID provided by the authors will be entered into the database. When the authors do not specify a sequence ID, these guidelines will be followed:

With an epitope formed by a continuous amino acid sequence, NCBI’s Protein BLAST is used to identify the sequence in the Swiss-Prot Database:

• Use the "Search for short, nearly exact matches" tool under PROTEIN BLAST links found at http://www.ncbi.nlm.nih.gov/BLAST/
• Enter the epitope sequence in the Search box and select <swissprot> under the Choose Database drop-down.
• BLAST the sequence and find the exact match.
• The Swiss-Prot ID from the GenBank file, found in the DBSOURCE tag will be entered into the database.
• When an exact match is not available, BLAST the sequence against "Non Redundant" databases by selecting "nr" under the Choose Database drop-down. When entering the GenBank ID, the GI number is used.

Epitopes from Display Libraries

When an epitope is identified through the use of a bacteriophage, baculovirus, or other randomized peptide library, it must be determined whether the phage-derived epitope sequence is homologous to a sequence from a biological source.

• If a natural homolog has been identified, then its sequence will be captured as the epitope.
• If a corresponding natural homolog was not identified in the reference, then select the [Source Antigen] as Phage Display from the source finder. <Phage display library> has the accession number of SRC-1627.

Carbohydrate Epitopes

The source of carbohydrate epitopes can be vague or confusing at times as carbohydrates may be present across many species. Always capture the source as described by the authors. If the authors mention multiple potential sources, assign the most generic source which captures all of the mentioned species. For example, if multiple strains of the same species are mentioned, curate the source for the generic species. If different species of the same family are mentioned, curate the source as the generic family.

Source Species/Strain

The species name from which the epitope sequence originates must be entered into the [Source Antigen] Source Species Taxonomy field. Often the epitope sequence originates from an organism such as a virus, bacterium, eukaryotic organism, or, less often, a plant. In order to enter the species into this field a Find function is used.

Use the Find button to access the Species/Virus Finder and type the name of the organism into the blank box followed by clicking on the Search Taxonomy button. Scroll through the entries for the genus and species name until reaching the appropriate strain. Double-click on the strain in order to enter it into the Source Species Taxonomy field.

Important Note: When the NCBI Species/Virus Finder provides both the source species and the strain, that option will be entered into the Source Species Taxonomy field. The strain must also be re-entered in the Strain field.

When the strain given by the authors is not found in the Species/Virus Finder, the genus and species name will be entered into the Source Species Taxonomy field and the strain name provided by the authors must be entered into the Strain field.

When the strain of the source species is not specifically mentioned in the reference and all uses of the source species are performed with a particular strain, the epitope may be attributed to that strain, provided the exact epitopic sequence is found in that strain. For example, when the authors describe a Clostridium botulinum epitope and do not mention which type it was derived from, but always immunize and assay with Type A, the epitope may be assigned the source species of Clostridium botulinumType A.

The same rules apply for species and strain when entered in the fields under the [Immunogen], [Antigen], and [Carrier] categories.

Important Note: Remember that the exact sequence of all epitopes must be found within the source antigen to which they are assigned. In the event that the epitopic sequence cannot be found within the source the authors describe, an IEDB Source ID may be assigned. Follow the guidelines below to obtain and use IEDB Source IDs.

When and How to use an IEDB Source ID

Natural sequence or non-natural sequence?

First it is important to identify if the epitope is natural or unnatural according to the authors. If the authors’ state the sequence was derived from a naturally occurring source (for ex, sequenced from a patient), then whatever source (GenBank, SwiProt, or IEDB) is used, it should have a natural source. If the authors state that the sequence was artificially created or is a lab induced mutation or analog of a wild type sequence, then the epitope should have NO Natural Source.

What if a sequence is not found in SwissProt/Genebank databases?

If the epitope is natural, first try to BLAST the sequence in SwissProt, if no EXACT matches are found, then try a Genebank BLAST.

Important Note: if the manuscript provides a long peptide sequence that does not match by BLAST, be sure to BLAST only the epitope sequence rather than the entire peptide sequence.

If still no match can be found by BLAST, then scrutinize where the mismatch occurs and confirm that the sequence you entered in the BLAST is the exact sequence specified in the manuscript. If there were no errors on your part, then determine if a typo was likely. Sometimes when a scanned doc is converted to a pdf, errors occur in sequences, placing unusual characters in the place of residues. An author may repeat a single aa unintentionally, for ex, the author states SIIINFEKL instead of SIINFEKL. With very obvious typos, for ex, the sequence is correct in several places in the manuscript, but entered with a typo in one location, it is ok to enter the correct, BLAST-matching sequence as the epitope. However, if there is any uncertainty, the author MUST be contacted in order to clarify the EXACT sequence used in the assays. Follow the guidelines for author contact and CTS use as applicable. If the authors fail to respond within 2 weeks and/or you cannot obtain a reliable source for the corrected sequences, proceed to assign a IEDB source ID.

IEDB Source IDs

IEDB source IDs are a list of molecules that are used as the source antigen when the SwissProt/GenBank databases do not have a matching source for a given epitope or when the source is non-proteinaceous.

Natural IEDB Source ID

If you are certain your sequence does not match anything by BLAST AND the author states the ENTIRE sequence being entered into the epitope sequence field is natural, then you should obtain or use an IEDB Source ID with a natural source. First check if an IEDB Source ID exists with the correct source antigen name for your case and that your species is part of the higher taxonomical category group the IEDB source is assigned to. If so, use this ID, and in YOUR epitope source field select the appropriate species and strain as provided in the reference. All uses of IEDB Source IDs are subject to review so be sure that you are using it correctly. If the particular ID you need is not present, then contact Laura Z. to obtain an IEDB Source ID and continue your curation with a place holder source ID and a note on your cover sheet stating that the ID must be changed before promoting the reference.

Unnatural IEDB Source ID

In the event an epitope sequence is unnatural, such as an artificial analog of a natural sequence, use an IEDB Source ID with an artificial source: SRC2066 IEDB No Natural Source Artificial Peptide/Protein is the most common selection. Important Note: When a natural sequence repeated an unnatural number of times is to be entered as the epitope sequence (because the natural sequence is not used in the curated assays), it should be entered as artificial & thus have no natural source.

SRC1627 Phage display library is to be used when an artificial peptide is obtained through panning of a Phage display library. The use of this IEDB source allows grouping of peptides that differ in the methodological basis of their discovery from analogs based upon a natural sequence.

Important Note: When a library is based upon a natural sequence, Phage display library (SRC1627) should be selected as the source.

Obtain an IEDB Source ID

To obtain an IEDB ID, email LZ (laura@liai.org) with the following information: PMID, epitope name and/or epitope sequence, location, & details regarding if & when the author was contacted. Laura will determine the correct IEDB Source ID to generate and notify you once it has been added.

Quasispecies/Minor Species

In the event the epitopic sequence is demonstrated to mutate over time, each new sequence represents a new epitope, antigen, or immunogen. If the new sequence matches a SwissProt/Genbank entry, that source may be used as the epitope source antigen. If the variant sequence does not match an existing sequence, an IEDB source should be assigned.

Epitope Positions in Source Antigen

The positions of the epitope provided by the authors may not match the positions given by Swiss-Prot (or GenBank ID).

The author specified positions are always recorded in the Epitope Starting Position and Epitope Ending Position fields.

The position numbers provided by Swiss-Prot will be recorded in the Epitope Swiss-Prot Positions field in the format starting position-ending position (for example, 120-129) and will be used only if there is a discrepancy between author reported and Swiss-Prot/GenBank positions. This discrepancy may be mentioned in the [Source Antigen]Comments field.

Be sure to enter the exact starting and ending positions as the authors state, even when these values contain errors. This field is the AUTHOR specified field, not the real positions.

Important Note: Repeating Epitopes
When exactly the same epitope sequence occurs at multiple positions in the same source antigen and reactivity is not demonstrated to be site specific, the positions of the first occurrence will be entered in the position fields with comments regarding the repetitive nature of the sequence entered in the epitope Comments field.

MHC Binding

Experimental data characterizing the interaction between an epitope and an MHC molecule is entered under the tab labeled MHC binding.

MHC Allele(s) that bind the epitope should be recorded exactly as specified by the authors. When a specified MHC Allele is not available for selection through the Allele Finder tool, please contact a senior curator or team member to add an allele to the finder.

Alleles that are mutated in the binding region in order to study the importance of residues in binding are not curated, but are commented on under the natural allele context (if present). Alleles that are mutated outside of the binding region are curated as the wild type allele. For example, a chimeric allele will be captured as the wild type of the binding portion.

Naturally occurring mutations in alleles are curated and should be added to the Allele finder if not present.

When both α- and β- chains of HLA class II alleles are specified in the reference, both of them will be recorded. Consult with EC members if you have any questions relating to the curation of MHC Alleles.

Important Note: All experimental MHC binding data given in a reference will be entered in the database irrespective of whether the qualitative assessment is positive or negative. This may not be bulk curated under any circumstances. TCR antagonism used in an MHC binding inhibition assay will be captured as an MHC binding context.

Important Note: In the curation of MHC binding assays, the exact peptide sequence that is tested MUST be entered as the epitope. There are no carriers possible in these assay types.

Qualitative Measurement

The Qualitative Measurement field is a required field and accepts only <Positive> or <Negative> as its value. The determination of positive or negative binding is established by the authors. The following guidelines are used to record qualitative measurement.

Case 1: When authors specify a qualitative assessment in the reference as either positive or negative, their assessment will be recorded in the database.

Case 2: When a qualitative assessment can be inferred from the information in the manuscript (threshold is provided), this assessment will be entered into the database.

Case 3: When no qualitative assessment is provided by the authors, this data will not be entered into the database.

MHC Ligand Elution

Only experimental data in which the authors elute peptides or ligands from a cell or purified receptors such as MHC is entered here. The peptide or ligand is then detected in the eluate through sequencing (Mass Spectrometry/Edman Degradation) or by a specific T cell with a known recognition of that peptide. These methods are used in order to demonstrate that cells will naturally process an antigen and present the epitope on the cell surface or bound to a relevant receptor. This specifically excludes epitopes given directly in the assay.

The processing of artificial antigens created in order to study processing are not curated. The processing of analogs created in order to study processing of the wild type sequence is also not captured. Processing of natural antigens should always be curated.

Important Note: When curating MHC Ligand Elution contexts in which the antigen presenting cells are incubated with the source species of the epitope, the protein from which the epitope was derived, or a fragment of the source antigen, the antigen type should not be <epitope>, but rather the larger antigen from which the cells derived the epitope should be selected (source protein or source species that was processed).

When the origin of the eluted peptide is not specifically known, the following guidelines apply:
-epitope of viral origin is eluted, the antigen type is <source species>
-epitope of known self origin is eluted, the antigen type is <source protein>
-epitope of unknown origin is eluted, the antigen type is <other>

Qualitative Measurement

Section (#Qualitative Measurement) applies.

T Cell Response

Minimum Criteria for Curation

Presence of Epitope

In general, polyclonal data generated without using the epitope as either the immunogen nor the antigen is not entered in the database for that specific epitope.

Table 5 serves as a guide to determine curatable contexts of polyclonal responses.

Table 5. Curatability of polyclonal T Cell/Antibody/B Cell Contexts

Immunogen		Antigen
		Epitope	Source Protein	Source Species	Other
	Epitope	Curate	Curate	Curate	Curate
	Source Protein	Curate	Do not curate*	Do not curate*	Do not curate*
	Source Species	Curate	Do not curate*	Do not curate*	Do not curate*
	Other	Curate	Do not curate*	Do not curate*	Do not curate*

Important Exception: When curating a deduced epitope, and neither the immunogen nor the antigen is the epitope, the assays performed to deduce the epitope should be curated and the antigen type should be either <Fragment of Source Antigen> or <Other>.

Curatability of Monoclonal Contexts (monoclonal antibodies and T cell clones): If the specificity of the monoclonal receptor is shown, known, or implied (for example, the T cell clone is generated by in vitro stimulation with the epitope) and the clone or mAb is tested for reactivity to source protein/species, or to a different antigen, this data can be curated for the epitope even though neither the immunogen nor the antigen is the epitope.

Pool of Peptides

When the antigen or immunogen is comprised of a pool of peptides and the result of the assay is negative, each peptide of the pool will be entered in the database (when provided by the reference). When an epitope is tested both as part of a pool and alone, those contexts may be bulked under the epitope used alone with a comment stating the same results were demonstrated through the use of a peptide pool.

When the response to a pool of peptides is positive, this data is generally not entered in the database but the de-convolution (testing of individual peptides within the pool) experiments are curatable, if available. However, there may be cases in which pools of overlapping peptides are used to define epitopic regions/domains without further de-convolution. If the peptide pool defines a continuous epitopic region/domain ≤ 50 amino acids, the data will be curated under the entire amino acid sequence encompassed by the peptide pool. The Epitope Comments field should explain that the epitope is actually comprised of a peptide pool.

TCR

TCR rearrangements V_β chain type, etc) are not captured in the current database structure. However, such data should be described in the Comments on Assay field under the appropriate T cell response.

T Cell Phenotype

T cell phenotype data (upregulation of co-stimulatory molecules like CD45) are not captured in the current database structure, but can be mentioned in the "Comments on Assay" field under the appropriate T cell response.

Tolerization Data

Data related to tolerization is not entered into the database. TCR antagonism is also not captured in the current database structure. This feature will be considered for future releases.

Antigen/Immunogen Fields

These fields clarify the relationships between the data entered in the Epitope Structure fields and the Antigen or Immunogen fields.

Antigen/Immunogen Type

Antigen and Immunogen Types are provided in a drop down menu composed of the following:

Epitope-This is to be used when the exact epitope structure is being used as the antigen or immunogen.

Important Exception: When a carrier or vector is used with the epitope structure, "Epitope" is selected. When the antigen or immunogen is the epitope presented in a different chemical type, for example DNA rather than peptide, "Epitope" is also selected.

Important Note: When a peptide linker is added to an epitope in order to link it to a carrier, the antigen or immunogen is to be curated as <Epitope>, the linker residues should be entered into the Comments on Assay or Immunization Comments fields.

Source Protein-This selection is made when the complete source antigen of the epitope is used as either the immunogen or the antigen.

Source Species-This is selected when the exact species and strain from which the epitope is derived is used in the assay.

Fragment of Source Antigen-This is to be used when any naturally occurring fragment of the source antigen that contains the epitopic sequence and is larger than the epitope structure is used. This selection is also used to capture the study of recognition of degradation analysis and enzyme or chemically treated fragments of the source antigen.

Important Note: When a number of fragments of the source antigen are tested, these fragments may be bulked into one assay context. Curate the fragment giving the best result and comment on the other fragments that were used.

Other Species/Strain-This is selected when a source species (virus, bacteria, etc) other than the exact species and strain from which the epitope was derived is used as either the immunogen or the antigen.

Other-Any immunogen or antigen that cannot be described by any of the previous choices will be labeled as "Other". Common examples of this type include peptides originating from species or strains other than those the epitope originated from and analog peptides.

BLANK The Immunogen Type may be left blank in certain circumstances. These will occur ONLY when the Immunization category is selected as: Unknown, Cancer, Phage Display, Autoimmune, and No Immunization.

Important Note: Remember that when the immunization procedure is unknown or uncertain, the Immunization Category is selected as <Unknown> and the Immunogen Type is left BLANK.

Multi-epitope construct -This selection is not to be used. It will be removed from the list at a future date.

Peptide containing the epitope-This selection is made when describing unnatural peptide contructs containing the epitope and additional residues, but no other functional units (other epitopes). This is only used to describe an unnatural construct. Author statements, curator discretion, and curatability rules should be used to determine whether to capture the Imm/Ag as Epitope with mention of additional residues in the Comments field if the additional residues warrant capturing the Imm/Ag as Peptide cont the Epitope. Different carriers linked to the same antigen or immunogen may be bulked. Curate the carrier giving the best response and comment on other carriers that were used.

Important Note: The curatability of peptides containing epitopes depends upon the sequence being recognized. For example, if the immunogen is the source antigen or the source species (containing the natural epitope sequence) and the antigen tested is the natural epitope sequence plus several unnatural residues, then the recognized & thus curated epitope is the sequence found in the natural source with the additional residues added for the purpose of the assay being either commented upon or captured as a peptide containing the epitope as further described below. However, if an artificial construct composed of a natural peptide sequence plus several unnatural residues is used as both the immunogen and the antigen, then the entire artificial construct must be captured as the epitope and be given No Natural Source. Additionally, an epitope source comment should reflect the natural source from which the epitope was derived.

Important Note: A natural peptide sequence should never be defined as Peptide containing epitope.

Important Note: When a peptide linker is added to an epitope in order to link it to a carrier, the antigen or immunogen is to be curated as <Epitope>, the linker residues should be entered into the Comments on Assay or Immunization Comments fields. Linker: A linker is a few residues used to LINK the epitope to another functional unit which may be another epitope, a MAP construct, a vector, etc. The linker is not entered as carrier. If the epitope is not linked to anything else, additional residues added to a natural epitope sequence are captured according to the above figure in either comments or captured as part of a Peptide Containing the Epitope.

Important Note: Regardless of the Imm/Ag Type selected and the particular use of the Carrier, Comments, and Adjuvant fields, no single component of the Imm/Ag should ever be repeated. That is, if certain residues are captured under the sequence of PeptContEpitope as selected for Imm/Ag, then those residues should not also be entered in the Carrier field. Likewise, residues captured in the Carrier field should not be entered in the Imm/Ag field. The same concept applies to the use of the Adjuvant field.

Important Note: In the curation of MHC binding assays, the exact peptide sequence that is tested MUST be entered as the epitope. There are no carriers possible in these assay types.

Antigen/Immunogen Chemical Type

Chemical Type DNA vs Pept/Protein

In all cases in which the immunogen or antigen is delivered in DNA form (plasmid DNA, naked DNA, DNA-coated beads/particles or DNA is delivered by an organism), the following guidelines apply: The Immunogen Type or Antigen Type fields will reflect the translated product (protein) of the DNA that was delivered in the immunization or used as the antigen in the assay, that is if the epitope was delivered in plasmid form, the Imm/Ag Type is <Epitope> and the Chemical Type field of the antigen or immunogen will be entered as <DNA>.

With the use of stably transfected cell lines expressing the epitope, the immunogen/antigen chemical type will be entered as the same as the epitope chemical type (protein). The cells expressing the epitope will be considered a carrier only when the cells expressing the epitope are used to immunize.

When a <multi-epitope construct> is used as an Antigen Type or Immunogen Type, only the corresponding [Antigen] or [Immunogen] Name fields must be completed. Details about backbone and/or other epitopes included in the construct are entered in the appropriate [Carrier] fields, including their sequences. This information is clarified in the appropriate Comment field.

Important Note: When the Antigen Type or Immunogen Type fields are entered as <Epitope>, <Source Protein> or <Source Species>, the remaining fields are autofilled. The curator should verify that all fields are accurate and may alter the chemical type and Imm/Ag Name if necessary.

Important Note: When a virus or cell expressing the epitope or antigen sequence is used as a vector, the information regarding the virus or cellular carrier is entered under the [Carrier] fields and the antigen/immunogen type is entered as the same type as the epitope.

Important Note: When the epitope or antigen is covalently bond to a vector, the information describing that structure is entered in the [Carrier]fields. When the epitope or antigen is not covalently bound to the vector, the information describing that vector is entered in the Formulation / Immunization fields.

Antigen Conformation Definition Field

This field describes the conformational type of the antigen used in a B cell assay as either native or non-native.

Native Select <Native> when the no alteration to the tertiary structure of the tested antigen has been made. Native conformation is commonly accepted as the biologically active form of the protein (or other chemical type). This also includes synthetic or recombinant peptides identified by the authors as having native conformation.

The following will be considered enough evidence for choosing Antigen Conformation Recognized = Native:

Antigen = Source Antigen

OR

Antigen = Source Species

AND

Assay Type = Neutralization Antibody dependent Cytotoxicity Assay Challenge Assay Cytopathic Effect Assay (CPE) Hemagglutination-Inhibition Calorimetry Colony Immunoblot Inhibition Assay

Non-native/unknown Select <Non-native/unknown> when the tested antigen is not in its native conformation. This would include short synthetic peptides and proteins that are deliberately denatured or denatured in their preparation. This value also includes antigens for which the physical nature of the antigen is neither stated by the author, nor decipherable from the paper (unknown).

Important Note: When authors’ state the conformation of the antigen is native, and it is reasonable to believe so, the conformation should be entered as Native, even when the above criteria are not met. Examples of such situations are when assembled viral particles or sporozoites are used in an ELISA and the authors’ specify recognition of native antigen.

Important Note: Assays having different antigen conformations may be bulk curated if the outcome is the same. Bulk curate under the native conformation.

MHC Fields

The following guidelines below are used to complete the MHC fields present under the T Cell Response and Naturally Processed Sections. Basically, if restriction is demonstrated for a T cell context, each restriction that is shown is curated separately as a new context with each allele listed once as the restricting allele. For example, if a CTL assay is done and restriction is known to be both HLA-A2 and HLA-A4, then two separate CTL assays will be curated, one with restriction of A2 and one with restriction of A4. Promiscuous binders are captured as multiple contexts if restriction is demonstrated in a manner to warrant the use of an evidence code. If the epitope is recognized by six alleles, for example, six contexts will be curated. When potential restriction is discussed without experimental evidence, restriction is not captured. The author's comments should be captured under assay comments and the MHC Types present field may be used. In cases where restriction is not fully demonstrated, but the MHC types of the responding population are known, these may be captured under MHC types present. The MHC types present field captures the common alleles shared by the responders among the group. Do not enter all MHC types tested, but rather, all MHC types shared by the population demonstrated to recognize the epitope. The purpose of this field is to add potential restriction information in cases where exact restriction is not yet known.

Important Notes:

• For Inbred animals with known fixed alleles (for example, inbred mouse strains BALB/C, C57BL/6), leave the MHC Types Present field under Immunized Species and MHC Types Present under Target Cell Species BLANK because these fields will be assumed standard and possibly autofilled in the future.
• If a restriction is not specified but the response is shown to be either a Class I or Class II response, then enter only the Class I alleles or the Class II alleles in the MHC Types Present field under the most appropriate category, [Immunized Species] or [Assay: Target Cells: Source Species], according to Figure 6 .
• In a population in which no allele(s) are shared by the population-leave the [Immunized Species] MHC Allele(s) Present field BLANK.
• If there are common allele(s) but none are specifically implicated as the restriction, then curate as MHC Types present.
• MHC restriction should be captured when the epitope has a known MHC binding motif and the antigen presenting cells express that allele.

MHC restriction Evidence Codes

The curator assigns MHC restriction based upon the following definitions and evidence codes. Only one evidence code may be selected at a time, therefore the code referring to the assay type which most narrowly defined the restriction of the epitope should be used. For example, if an antibody blocking assay was used to narrow the restriction to Class I and an assay using mismatched APC was used to define which Class I allele defines the restriction, the evidence code selected should be mismatched MHC molecules.

1)T cell assay indicating MHC restriction

• T cell assay -Mismatched MHC molecules

Use of APCs expressing differing MHC molecules. The assignment of MHC restriction is based upon the use of a T cell assay in which animals or cells of differing MHC types are used to demonstrate MHC restriction.

• T cell assay -MHC subset identification

The assignment of MHC restriction is based upon the use of a T cell assay in which antibody is used to block a MHC molecule or MHC molecule subset in order to demonstrate MHC restriction.

• T cell assay -Single MHC type present

The assignment of MHC restriction is based upon only one MHC type being present in a T cell assay. For example, a CTL assay with APCs expressing a single type of MHC molecule.

• T cell assay -Biological process measured

Biological process measured-applies only to class restriction. For example, assignment of Class I restriction for an epitope used in a CTL assay. This is applied only when clear indication of restriction is present.

• T cell assay -T cell subset identification

The assignment of MHC restriction is based upon the use of a T cell assay in which antibody is used to block a subset of T cells, a subset of T cells is depleted, or a pure T cell population is used in order to demonstrate MHC class I or Class II restriction.

2)MHC binding assay The assignment of MHC restriction is based upon the results of a MHC binding assay.

3)MHC binding prediction The assignment of MHC restriction is based upon the use of a binding prediction based upon the sequence or structure of the epitope without experimental assay.

4)Statistical association The assignment of MHC restriction is based upon associations of the study population’s MHC expression and recognition of the epitope.

5)Cited reference The assignment of MHC restriction is based upon information provided in references cited by the author.

Effector cell assignment is made following these guidelines:

1.Phenotype identification- Direct demonstration of the effector cell phenotype will be used to assign effector cell type. For example, CD8+ staining of the population producing IFNg.

2.Cell Isolation –Isolation or purification procedures will be used to identify the cell type of the effector cells present in the assay. For example, the use of a cell population after CD8 depletion would be identified as CD4+ T cells.

3.Biological process measured-The response measured by the assay type will not be used to identify the cell type of the effector cells. For example, measurement of proliferation may be an indicator of CD4+ T cells, however, if splenocytes were used in the assay, the effector cell type should be entered as splenocytes.

4.MHC Restriction –MHC restriction of the epitope will not be used to assign the cell type of the effector cells used in the assay. That is, if PBMC are used in an assay utilizing a Class II epitope, PBMC will be entered into the effector cell field. However, assignment of specific MHC restriction or restriction to the level of Class I or Class II should be performed whenever possible and may be attributed to the assay type when the authors state or imply such.

Immunization

Immunization Category

This field describes all exposures of the immune cells under study both in vivo and in vitro from initial exposure until the time of the assay. When subjects are vaccinated, cells are infected or stimulated in vitro, the selection will be <Administration>. <Administration> implies that the initial contact of the immune system or cells is by deliberate and controlled exposure. All other Immunization Category selections imply that the initial exposure to an antigen is by natural biological processes, e.g. infection, cancer, allergens, or autoimmunity. The possible selections are described in detail below.

Important Note: The Immunization Category field is independent from the Disease state and the [Immunogen] fields.

<Administration>
The immunogen was administered for the purpose of immunization or stimulation either in vitro or in vivo.

Important Note: Select <Administration> only when the Immunogen is known. Do not select <Administration> when the Immunogen is unknown, even when you can safely assume administration of the immunogen was performed. All selections of <Administration> must have the Immunogen field filled in.

<Allergy>
Subjects have a naturally occurring allergy against the immunogen (allergen) prior to the study.

<Allergy plus restimulation>
Cells are taken from individuals with a prior allergy and restimulated In vitro.

<Autoimmune (No Administered Immunization)>
The immunogen (if known) originates from the host, such as with effector cells believed to be specifically reacting to a self antigen when derived from a subject with an autoimmune disease.

<Autoimmune plus restimulation>
Cells are taken from a subject with an autoimmune disease and restimulated in vitro.

<Cancer (No Administered Immunization)>
Effector cells are taken from a cancer subject and are believed to specifically recognize cancer antigens.

<Cancer plus restimulation>
In vitro restimulation is performed on cells obtained from individuals with cancer.

<Natural Infection or Exposure>
Subjects are naturally infected prior to the study.

Assumptions: For certain ubiquitous pathogens such as Influenza, CMV, EBV and Candida, all individuals are presumed to have natural exposure to the source species (when not clearly stated by the reference). Similarly, PPD positive individuals are assumed to be naturally exposed to mTB pathogen when not specified in the reference. When curating samples taken from endemic areas, the immunogen may be assumed based upon location, for example, in the case of PBMC collected from healthy donors living in a malaria endemic region, the immunogen will be captured as P falciparum.

<Natural Infection or Exposure plus restimulation>
In vitro restimulation was performed on cells taken from individuals who were naturally infected or exposed.

<Phage Display (No Immunization)>
Antibodies are obtained through the use of a phage display library. In this situation, all [Immunization] fields will be left blank.

<Unknown>
The immunization category is not specified in the reference. Select <Unknown> whenever the Immunogen is unknown. This can occur when an antibody is purchased from an outside lab or commercial company or a previously described cell line is used. All available information will be mentioned in the Comments on Immunization field.

Important Note: In cases with no immunization such as cancer, autoimmunity, or unknown natural exposure with additional in vitro restimulation, the restimulating antigen is entered as the immunogen.

Immunized Species

The species and strain or ethnicity of the immunized species is captured in this field. Different species cannot be bulked. Each new immunized species is curated as a new context. Different strains may be bulked in certain situations. When the outcomes of the assays are the same for B cell assays, different strains of mice may be bulked. All of the different strains should be entered in this field, separated by commas. Different strains may be bulked in T cell contexts if the outcome is the same and the MHC alleles are the same. The curator should use discretion and author statements to determine when different strains of animals should not be bulk curated.

Transgenic Mice

The following guidelines apply when the immunized species is a transgenic strain of mice.

• The Species field will be "Mus musculus"
• The Strain field will specify that it is a transgenic mouse (BALB/C A2 Transgenic)
• Human alleles will be recorded in MHC Types Present field when the transgene contains an MHC allele; otherwise the guidelines in (#MHC Fields) will be followed.

Ethnicity

Ethnicity can be defined as "of or relating to large groups of people classed according to common racial, national, tribal, religious, linguistic, or cultural origin or background".

Ethnicity will be entered in the Strain/Ethnicity/Population field when the ethnicity is clearly stated in the text and it can be found in the "Population" or "Population Area" lists at:

http://www.ncbi.nlm.nih.gov/projects/mhc/ihwg.cgi?cmd=PRJOV&ID=9

Information regarding the ethnicity will be entered in the Comments on Immunization field when the ethnicity is not clearly specified in the text, does not appear in the "Population" list, or a geographic region is used to describe the origin of the patients involved (geographic location does not necessarily correlate with ethnicity).

Disease State

The Disease State field reflects the disease state of the individual(s) used in the context. When multiple diseases are reported in the reference, the one disease which is most appropriate to the context will be entered while the others will be noted in the Comments on Immunization field.

Diseases which are the result of an administered immunogen are only to be captured in the Disease State field when evidence of disease is demonstrated (clinical symptoms, pathological evidence, author stated, etc) and/or are relevant to the assay being captured.

Assumptions: For certain ubiquitous pathogens such as Influenza, CMV, EBV and Candida, all individuals are presumed to have natural exposure to the source species (when not clearly stated by the reference). Similarly, PPD positive individuals are assumed to be naturally exposed to mTB pathogen when not specified in the reference.

Important Note: When curating samples taken from endemic areas, the immunogen may be assumed based upon location, however, the the Disease State field is not used unless the author's indicate that the population has or had a specific disease. For example, in the case of PBMC collected from healthy donors living in a malaria endemic region, the disease state field will be left empty and the immunogen will be captured as P falciparum.

Disease Stage

The Disease Stage field reflects the stage of the disease at the time the experimental data was generated. The Disease Stage may be described as:

<Acute>
A short-term infection or disease characterized by a dramatic onset and rapid recovery is recorded as <Acute>. Primary infections fall under this category.

<Chronic>
A long-term infection or illness and partial remission is recorded as <Chronic>.

<Post>
An illness a subject has recovered from is recorded as <Post>. Latent (potentially existing but not presently evident or realized) and remission (a period during which symptoms of disease disappear [complete remission]) are recorded as <Post>. Note that partial remission will be recorded as <Chronic>.

<Other>
Any disease stage that cannot be classified under <Acute>, <Chronic> or <Post> will be classified under <Other>. Cancer stages and household contacts will be recorded as <Other>.

<Unknown>
When the disease stage is not clearly specified or unavailable in the reference, it will be recorded as <Unknown>. The main distinction between <Other> and <Unknown> is that <Other> specifies a disease stage mentioned in the reference, but not classifiable as <Acute>, <Chronic>, or <Post> while <Unknown> specifies a disease stage which is not explicitly mentioned.

Recording Multiple Immunizations

When there are multiple immunizations reported (possibly involving a number of different immunogens), only the first immunization and immunogen used is captured in the [Immunization] fields with the subsequent Immunizations being recorded in the Comments on Immunization field.

Important Note: When several immunization procedures, schedules, and doses are used, these may be bulked into one assay context. Curate the procedure giving the best results or the one most often used and comment on the other procedures.

Number of immunizations
This field describes the entire immunization schedule including dosing and formulations.

In vivo Prime / Boost
When multiple immunizations with a number of different immunogens is used, the first immunogen is recorded in the Immunogen field with information regarding subsequent immunizations recorded in Comments on Immunization.
It is very important to enter this information in the Comments on Immunization field because the future database design may be capable of capturing this information separately.

In vitro restimulation
Effector cells may be taken from a host and restimulated in vitro prior to the assay. This restimulation may be performed with the epitope itself or an antigen containing the epitope in order to amplify an existing response to detectable levels in an assay.
When cells from an immunized (exposed) subject are washed after restimulation and before antigen is added to measure a T cell or B cell/antibody response, this is considered an in vitro restimulation and the in vitro restimulation information will be recorded in the In Vitro Immunization/Restimulation field. The in vivo immunization fields will reflect the in vivo immunization or exposure process. When cells are taken from an immunized subject and directly assayed in the presence of antigen, this is not considered restimulation.
When cells are derived from a naïve subject and first encounter the antigen in vitro, this is considered the immunization process and is not considered an in vitro restimulation.

Important Note: Growing dendritic cells in vitro, prior to use in a T cell or B cell restimulation, involves stimulation of the dendritic cell culture. This procedure is not considered part of the in vitro restimulation. Record this information in the Comments on Immunization field.

Carrier

A carrier is usually a molecule covalently linked to an antigen in order to increase its antigenicity. When the carrier is attached to an epitope’s molecular structure, fragment of protein, the source protein, or the epitope’s source species, the immunogen will be the molecule attached to the carrier and the information describing the carrier will be entered in the fields In the [Carrier] section. Different carriers linked to the same antigen or immunogen may be bulked. Curate the carrier giving the best response and comment on other carriers that were used.

The most significant distinction between a Carrier, a Formulation and an Adjuvant is that a carrier is covalently linked to the immunogen molecule, whereas adjuvants are substances that are administered with the immunogen in order to enhance the immune response and are not covalently linked to the immunogen. Chemicals other than adjuvants that are mixed with the immunogen are described in the Formulation Form field.

Until further changes are made to the IEDB ontology, vectors used in the context of a T or B cell response will be captured in the [Carrier] fields. This includes bacteria, virus (other than the source species) or cells when used in the delivery of an epitope.

Important Note: When a peptide linker is added to an epitope in order to link it to a carrier, the antigen or immunogen is to be curated as <Epitope>, the linker residues should be entered into the Assay Comments or Immunization Comments fields.

Formulation

The [In Vivo Immunization] Formulation field is a free-text field used to describe relevant information regarding the delivery of the immunogen that is not recorded under the [Carrier] or [Adjuvant] fields. Substances which are neither covalently linked nor clearly specified as an adjuvant that are mixed with the immunogen in order to enhance the response may be entered in the [In Vivo Immunization] Formulation field.

Important Note: When several immunization procedures, schedules, and doses are used, these may be bulked into one assay context. Curate the procedure giving the best results or the one most often used and comment on the other procedures. Different adjuvants should also be bulked. Curate the adjuvant giving the best results or the one used most often and comment on other adjuvants that were used.

Cell Types

The Cell types are chosen from a drop-down list. This list contains both histological cell types as well as the names of immortalized cell lines. Additional cell types or names may be added to this list through the database administrator.

Certain cell types or names may be represented as alternative titles or acronyms in a reference. Consult a senior curator or EC member regarding discrepancies or confusion prior to requesting additional values be added to the current list.

Important Note: When different APC or effector cells are used in the same assay type, they may be bulked. Curate the APC giving the best response or the APC type that was used most often in the reference. For multiple effector cell types, different T cell lines and clones may be bulked as well as PBMC and select T cell populations (CD4+, for example). When bulking effector cell types, be sure to capture the most relevant data and never bulk different immunogens or different immunized host species.

Hybridoma

Hybridomas are entered into the database under the following guidelines:

• The Tissue Type field is left BLANK
• The Cell Type field is <Hybridoma>
• The Origin field is <Cloned / Long term restimulated / cell line>
• A brief description describing the hybridoma is entered in the Comments on Assay field

Origin of Effector and Antigen Presenting/Target Cells

The Origin field describes the origination of the effector or APC/ target cells.

<Cloned / Long term restimulated / cell line>
Cell lines or clones obtained through long term restimulation are designated as <Cloned/Long term restimulated/cell line>. Operationally we consider T-cell populations polyclonal that have no or limited in vitro stimulation. Repeated T-cell stimulations in vitro and long term cell lines will be considered monoclonal and designated as a T-cell line. Oligoclonal T-cell populations that have received many rounds of in vitro stimulation are presumed monoclonal unless otherwise stated by the authors.

<Restimulated in vitro prior to assay>
Cells taken from an animal and stimulated in vitro with the antigen for a certain period, after which cells are washed and antigen is added again for the purpose of assay, are designated as <Restimulated in vitro prior to assay>.

<Directly assayed (in vitro)>
Cells taken from an animal with antigen added only for the purpose of the assay without any prior in vitro stimulation are labeled as <Directly assayed (in vitro)>.

<Assayed in vivo>
Cells that are assayed in vivo are designated <Assayed in vivo>.

<Other>
Cells that are assayed by other means, which cannot be captured by any of the above choices, are designated as <Other>.

Assay

Effector Cells

Effector cells may be referred to as CD4+, T helper cells, CD8+, or CTLs even though a broader cell population was used in the assay. When there is no clear indication that the cells were purified from a broader population (for example PBMC, Spleens, etc), the effector cells will be entered as belonging to the broader population. When a specific T cell population is tested using a tetramer assay, specify the effector cells as either CD4 or CD8 cells.

Important Note: Effector cell assignment is made following these guidelines:

1.Phenotype identification- Direct demonstration of the effector cell phenotype will be used to assign effector cell type. For example, CD8+ staining of the population producing IFNg.

2.Cell Isolation –Isolation or purification procedures will be used to identify the cell type of the effector cells present in the assay. For example, the use of a cell population after CD8 depletion would be identified as CD4+ T cells.

3.Biological process measured-The response measured by the assay type will not be used to identify the cell type of the effector cells. For example, measurement of proliferation may be an indicator of CD4+ T cells, however, if splenocytes were used in the assay, the effector cell type should be entered as splenocytes.

4.MHC Restriction –MHC restriction of the epitope will not be used to assign the cell type of the effector cells used in the assay. That is, if PBMC are used in an assay utilizing a Class II epitope, PBMC will be entered into the effector cell field. However, assignment of specific MHC restriction or restriction to the level of Class I or Class II should be performed whenever possible and may be attributed to the assay type when the authors state or imply such.

T Cell Clone Name

The name of T cell clones, when specified by the authors, is entered in the TCR Name field present in the [Effector Cells] section only when there is a clear reason to do so. For example, the authors describe recognition or lack thereof by certain clones and specifically discuss the unusual reactivity of the clones. Differing fine specificity of clones does not warrant entering the clone names. All clones generated through immunization with the same immunogen may be bulked, regardless of the restimulating antigen, carriers, or adjuvants.

APC/Target Cell Species

The Assay: Target Cells: Source Species section captures information regarding the species of the target cells (antigen presenting cells). <Y> is entered in the Autologous Cells field when the target cells are derived from the same individual from whom the effector cells were derived.

When target cells (or antigen presenting cells) are obtained from syngeneic individuals, they will be considered autologous. Syngeneic means genetically identical members of the same species. When syngeneic cells are used, a comment will mention this in the Comments on Assay field.

Important Note: The use of different APC for the same assay type may be bulked. Capture the APC type giving the best result or used most often and comment on all other APC used.

Special Culture Conditions

Information recorded in the Assay: Special Culture Conditions field MUST reflect Information that is both special and pertains to culture conditions. This field is only used to record significant deviations from standard protocols that are critical for the measured response. For example, adding lipopolysaccharide to induce maturation of dendritic cells in culture. Do not enter comments about immunization or assay in this field.

Qualitative Measurement

Section (#Qualitative Measurement) applies.

Quantitative Measurement

Curating numerical values has low priority except In MHC and antibody binding assays measuring IC₅₀ or affinity constant values. Conclusions about relative effects and the magnitude of responses in different contexts should be summarized in the comments.

Enhancement Assays

In enhancement assays, a T cell epitope is used to enhance either a T cell response or an antibody response. There are two assay types: T cell Assay-Enhancement of antibody response and T Cell Assay-Enhancement of T cell response. An enhancement assay is curated under the T cell epitope which is responsible for the enhancement, ONLY when the manuscript demonstrates that the T cell epitope is responsible for the response, that is, the response MUST be tested with and without the presence of the T cell epitope.

Use the following guidelines to curate these assays:

The immunogen is always the T cell epitope. Immunogen Type =<Epitope>.

The antigen is the entity to which the response is enhanced. For example, with enhancement of the antibody response to the B2 epitope, the antigen is the B2 epitope. Antigen Type =<Other>. With enhancement of the antibody response to HBV core protein, the antigen is HBV core protein. With enhancement of the T cell response to the T4 epitope, T4 epitope is the antigen. Antigen Type =<Other>.

Important Note: When a T cell epitope enhances the response to a number of B cell epitopes, that data may be bulked with the antigen entered as a representative B cell epitope and the other epitopes commented upon in assay comments. Enhancement of the response to multiple T cell epitopes may also be bulked.

B Cell Response

Minimum Criteria for Curation

Section (#Minimum Criteria for Curation) applies.

Special Note: All assays generating K on (Ka [M^-1 s^-1]), K off (Kd [s^-1]), and KD values (Enzyme-Linked Immuno Sorbent Assay (ELISA) or Surface Plasmon Resonance (SPR)) are always to be curated as separate contexts.

Antigen/Immunogen Fields

Section (#Antigen/Immunogen Fields) applies.

Immunization

All sections under (#Immunization) apply to B cell/antibody contexts.

Antibody Display Libraries

When antibodies originate from an antibody display library (bacteriophage or other construct), the Immunization field should reflect the status of the individuals from which the library was derived. <No immunization> is selected with the use of naïve or synthetic libraries. See Appendix (section (#Displayed Antibodies)) for more details on Displayed libraries.

Passive Immunization

Passive immunization data is not currently under the scope of the Immune Epitope Database, however, this type of data may be included in the future. Passive immunization is accomplished through the transfer of antibodies or immune cells from an Immunized subject (naturally or by vaccination) to a naïve individual.

Assay

Materials Assayed

This field clarifies the origin and purification status of the antibody used. Values such as serum, purified immunoglobulin, etc are available through a pull down menu.

In the event the antibody binding domain (Fab, Fv, VHH, etc) is artificially displayed on a construct or phage, <Displayed Ab(s)> is selected.

Special Note: With antibody originally selected from a library and subsequently expressed without a carrier, the Materials assayed will be <purified immunoglobulin> and the library origin will be reflected in the Antibody type field as <Display library>. See the Appendix (section (#Displayed Antibodies)) for more details on display libraries.

Recording Chimeric Antibodies

Chimeric antibodies are generated from more than one species. Currently the Source Species field will not accept two values. Use the Find function to search for "Chimeric" in the Antibody Species field to enter <Chimeric (more than one source)> in this field. A description of the multiple species the antibody was derived from is included in the Comments on Assay field.

Antibody Isotype

When the isotype of the antibody is not specified in the reference, but a known secondary antibody is used, the isotype of the primary antibody may be inferred from the secondary antibody used in the assay. In most of the cases, the Chain 1 Isotype field will capture the heavy chain and the Chain 2 Isotype field will capture the light chain.

Different isotypes may be bulked when the outcome is the same and the distinction between them is not significant. Always comment on all of the isotypes studied. If the authors discuss the different isotypes and/or it is a significant point of the paper, curate the isotypes as separate assays.

Special Culture Conditions

Section (#Special Culture Conditions) applies.

Qualitative Measurements

Section (#Qualitative Measurement) applies.

Quantitative Measurements

Section (#Quantitative Measurement) applies.

Individual Assays

Tetramer/Multimer Assays

In MHC Tetramer/Multimer staining assays, there are neither Target Cells nor Antigen-Presenting Cells. Therefore, leave the fields under these headings blank:

Antigen Presentation - Antigen Presenting Cells: Tissue Type, Cell Type, Origin.

However, the MHC restriction of the tetramer must be entered in [Antigen Presenting Cells: MHC] MHC Allele(s) field.

The information describing the effector cells is entered as usual along with the details of immunization.

Challenge Assays

In accordance with rule (#Presence of Epitope), challenge assays in which the immunization is performed with the epitope as the immunogen are the only challenge assays included in the database. The virus used as the challenge will be recorded as the antigen under either a T cell or B cell context, depending on other information available for the epitope.

Crystal Structure/NMR Assays

3-D structural data is the domain of experts at SDSC. It is advised that all references containing 3D-structural data should be discussed with a senior curator to determine the curatability.

Western Blots

When a whole cell lysate is studied by western blot, the Antigen Type selected is according to the level of purification or separation performed. Whole cell lysate is to be captured as <source species> rather than source protein. If further separations or purifications are performed such as immunoprecipitation or fractionation studies prior to being run on the gel, the Antigen Type will be <source antigen>. The concept is that pools of protein are captured as protein while heterogeneous pools of surface and cytoplasmic molecules are captured as species. Refer to a senior curator or EC member for difficult scenarios. Comments on Assay should be used to express any specific reactivity to particular proteins/bands observed on the gel and author statements regarding reactivity.

Important Note: In the event a cell or organism is constructed to express the protein of interest and the whole cell lysate of the engineered cell/organism is run on the gel, the Antigen Type will be <source antigen>.

Important Note: Prion proteins studied by western blot will be captured as <source antigen>.

Competition/Inhibition Assays

When curating inhibition/competition assays, the antigen is always the competitor. However, when the entity whose binding is being inhibited is not captured in any other context, this interaction should also be curated in a separate assay as a direct binding/recognition assay when that interaction is curatable. Thus, the competitor will be the antigen in the competition assay and the inhibited antigen will be captured as the antigen in a binding assay. Note that this only applies if that binding is not captured elsewhere and is curatable.

Direct binding and competition assays with the same antigen, immunogen, and outcome should be bulk curated under the competition assay.

Bulk Curation

Bulk Curation refers to capturing multiple data points under one context record. Obviously this strategy is not ideal for all context records, but applies only to certain scenarios. When performing bulk curation, the overriding goal is to capture the authors’ conclusions. Different antigens, immunogens, and host species are never bulk curated. Assays giving different outcomes are never bulk curated. Different assays which measure the same type of response may be bulked curated. For example, different assay types measuring the binding of a mAb to the same antigen (WB & ELISA) can be bulk curated. Do not bulk curate assays which measure different responses such as the expression of different cytokines.

Common Cases for Bulk Curation

The following scenarios are commonly encountered cases for bulk curation and should always be curated in the same manner.

Mapping an Epitope

Truncation

When peptides are tested in the context of epitope mapping or minimal epitope analysis (residues are truncated to discover the minimal epitope) only the minimal epitope is entered. The other peptides that were part of the minimal epitope analysis are not curated. For class I epitopes only sequences of greater than or equal to 7aa are considered epitopes. Shorter sequences deduced from truncation analysis and not assayed directly as a peptide are considered "core residues" of an epitope of length defined by the shortest peptide used in the truncation experiment. In the figure below (Figure 7 , only the epitope designated as D4-NS3 _221-232 is curated.

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Protein Scan/Overlapping Peptides

Overlapping peptides tested as a part of a protein scan are recorded as separate epitopes and are not bulk curated irrespective of their qualitative assessment (negative or positive). An example of this type of situation is shown in the figure below (Figure 8 ) pasted from reference with PubMed ID 15356154. All of the peptides in the figure are entered into the database as separate epitopes.

Important Note: These guidelines apply to both polyclonal and monoclonal responses.

Important Note: Epitope mapping by truncation rule (#Truncation) takes precedence over Protein Scan/Overlapping Peptides rule (#Protein Scan/Overlapping Peptides). For example if overlapping peptide scan is shown, followed by epitope mapping by truncation, the epitope mapping rule (#Truncation) should be followed and thus only the minimal epitope is curated.

Curating Contexts with Multiple Subjects

When the same assay is performed on samples derived from various subjects, the data will be entered as a single record. The main conclusion of the entry should be focused on an epitope-specific result rather than on the differences between various samples. In these cases the [Immunization] and [Assay] fields will capture the common information for all of the subjects, such as common MHC alleles present in the donor population. The Number of Subjects Immunized and Number of Subjects Responded fields are used to reflect the use of various subjects.

Mapping the MHC Restriction of an Epitope

Often the exact MHC restriction of an epitope is determined by using various APC/target cells with different alleles present. This type of data will not be captured as separate contexts; instead the MHC restriction will be inferred and entered for other contexts in the same reference.

For example, in Figure 9 (pasted from reference with PubMed ID: 12023403) below, MHC restriction of a cell line is determined by testing multiple cell lines expressing different MHC alleles. In the same reference, the specificity of this dengue virus CD8+ CTL line was identified as a linear class I epitope. Other data in the reference is presented for the epitope along with a MHC restriction analysis. Figure 9 is not curated as a separate context, rather the MHC restriction will be inferred for other contexts and curated for the epitope identified from the dengue virus CD8+ CTL line.

The same applies to all other assays solely performed in order to determine MHC restriction, such as the use of MHC specific antibodies.

Dose-Response Curves

Whenever a series of assays varies by only one variable, such as the dose of a peptide or adjuvant or the time of exposure, only the conditions giving the highest response will be curated with the other conditions noted in a comment.

T Cell Clones

Responses from multiple T cell clones will be bulk curated as one context when the T cell clones recognize the same epitope. For example, the data in Figure 10 (pasted from reference with PubMed ID: 15033572) will be bulk curated and entered as one context for each epitope.

Panel of Monoclonal Antibodies

When a panel of monoclonal antibodies is used to characterize binding to an antigen, the antibodies demonstrating comparable reactivities in an assay are bulk curated by entering all antibody names (comma delimited) in the Antibody Name field. A separate assay entry must be used for antibodies showing differing reactivities. When multiple classes or isotypes of antibody are encountered during bulk curation, the most relevant or most common antibody class/isotype used in the assay should captured in the drop-down menus for Antibody: Chain 1 isotype (and Chain 2 isotype, if it is known). The remaining information may be captured in the Comments on Assay field.

Decision Scheme for Bulk Curation

This section is currently under review. Use cross reactivity guidelines and relevance to the reference when applying novelty points. New guidelines may appear in the next version of the manual.

When section (#Common Cases for Bulk Curation) (common cases for bulk curation) does not apply with multiple contexts that can be either curated separately or bulked together, follow the guidelines below:

Once one of the contexts is entered into the database a determination is made regarding the novelty of the remaining data. Points are given based upon the table below. When the sum of novelty points is greater than 2, the data will be entered as separate contexts. When the sum is 2 or less, the data will be bulk curated with a comment to capture the remaining information. Use Table 6 to determine the novelty points.

Table 6. Determining contexts to curate – Novelty Points

Difference between contexts	Novelty Points
New Epitope Molecular Structure, Analog or Mimotope	3
New Epitope Source Species / Protein	2
New immunized species (host)	3
Antigen Type / Immunogen Type switches between peptide and "Source Protein" or "Source Species"	3
New immunized strain for T cell response	3
New immunized strain for B cell response	2
New qualitative outcome	2
New natural antigen / immunogen	2
New type of response measured	2
Difference in any other field except comment field	1

Negative Data

Monoclonal Receptors

Negative Immunogenicity data is not curated when the effector receptor (T cell or B cell/antibody) tested is monoclonal and there is positive data for the same receptor available. This is due to the implication that monoclonal receptors are highly specific.

Important Exception: Negative monoclonal data will be captured when the response is expected to be positive, but is negative and this lack of response is of interest.

Important Note: Negative data from T cell lines believed to be monoclonal will not be curated. Multiple passages and restimulation procedures are necessary to imply clonality of T cell lines. Short term cell lines will not be treated as monoclonal (unless otherwise demonstrated by the authors) and thus negative data generated by their use will be curated.

MHC Binding Data

Both positive and negative MHC binding data is always recorded. All MHC binding data is curated even when it is used to determine a minimal epitope.

Testing a Pool of Peptides

Section (#Pool of Peptides) applies. Negative data generated from pools of peptides is curated. Data from submissions like epitope discovery contracts should be encouraged to include quantitative information from testing pools of peptides, while for literature and patent curation, quantitative information is usually not recorded except for binding constants.

Miscellaneous

Handling Special Characters

Special characters (Greek letters, symbols) entered in database fields are recorded using special codes. A complete list of special characters and its respective code to be recorded can be found at

http://www.visibone.com/htmlref/char/ceralpha.htm

For example, if a particular word contains ã or æ or λ, you will need to type the corresponding code ã or æ or λ

Superscripts and subscripts require a special format for input. To enter a subscript, type _''text''. Likewise for superscripts, type ^text. For more assistance in capturing special characters, please contact a senior curator.

Data Not Shown

Data not shown in the manuscript will not be entered into the database as a separate context, but may be commented on under similar contexts. When authors discuss results without actually showing the data in that particular reference, one may infer that the same protocols described in the reference were used to obtain those results.

• When data is given as supplementary data, it will be curated.
• When there is data provided in the reference, but not discussed in the text by the authors, this data will not be curated.

Appendix

Displayed Antibodies

• The cDNA of an antibody is cloned and expressed on the surface of macromolecular complexes and individual binders are subsequently selected. The cDNA is generally attached to the macromolecular complex to allow for rapid determination of the sequence of the positive clones. This technology is an alternative to the classical monoclonal antibody method using hybridoma generation from secreting clones.
• The most common library type is phage display, but other constructs, such as ribosome or yeast display, may be used.
• The antibody cDNA which is displayed may originate from either naïve or immunized individuals. Non-human libraries are usually immune and human libraries are usually from naïve or naturally exposed individuals, but there are exceptions. The Immunization field should reflect this status when provided by the reference. If the library is derived from a naive subject(s), the Immunization Category should be <Phage Display (No immunization)>. Immune libraries have increased frequency of binders and higher affinity of binding, but naïve libraries are often a source of binders of varied specificities and may be commercially available. Synthetic libraries consist of artificially generated antibody sequences and require a comment in the Comments on Immunization field to clarify what antibodies were used.
• The type of assays to be entered as contexts are those typically included in other antibody contexts, such as epitope characterization by binding of particular clones. Selection assays (immuno-panning) is not included in the database, unless that data is highly relevant to the subsequent analysis. If needed, this assay type is present under B cell response.
• The antibody displayed is typically not the entire molecule but the binding fragment. Depending on the cloning procedure, Fab, Fv, scFv or Fab’ fragments may be displayed. This information is to be entered in the Immunoglobulin Domain field. Camelids (camel, dromedary, llama, etc) have a particular IgG isotype devoid of light chains. The binding fragment (variable region of this IgG) is a unique polypeptidic chain named VHH.
• When the displayed antibody is the material assayed, the Materials Assayed field should be <Displayed Ab(s)> and Antibody Type should be <Display library>. The monoclonoal or polyclonal nature of the antibody will be entered in the Assay Comments field, while the Immunization Comments (if any) should include that the immunization was followed by display library construction.
• When a recombinant antibody is subsequently expressed in the absence of the displaying particle, the Materials Assayed field should be <Purified immunoglobulin>, the Immunoglobulin Domain field should reflect the fragment used and the Antibody Type field should be <Display library>. As before, the polyclonal or monoclonal nature will be entered in the Assay Comments field with any immunization comments including that the immunization was followed by display library construction.

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3D Structural Data

The IEDB includes 3D-structural data describing complexes of epitopes with immune receptor(s): antibodies (or antibody fragments including the variable region), MHC molecules and/or the T cell receptor (or fragments including the antigen recognizing domains).

Exclusions

• Structures in which the antibody or TCR binds to the epitope outside of the variable antigen-binding region are not curated.
• Structures in which the MHC molecule binds the epitope outside of the recognition groove are not curated

3D data is captured in the IEDB by structural experts at SDSC when:

• The structure is obtained by an experimental method.
• 3D-structure is available in the Protein Data Bank (http://www.rcsb.org)

Each novel structure is captured only once as a context of the epitope which is bound by the immune receptor. That is if an epitope is bound by an antibody and an MHC molecule, two assays will be captured, one for each structure.

If there are different structures describing different contact residues between the epitope and a receptor, each set of differing contact residues is captured as a separate assay.

Standard Abbreviations

The table below (Table 7 ) shows the list of standard abbreviations consolidated from different immunological journals.

Table 7. Consolidated list of standard abbreviations.

#	Abbreviation	Expansion
1	14C, 3H, 32P, etc.	isotopes
2	2D/3D	two dimensional/three dimensional
3	2-ME	2-mercaptoethanol
4	Å	angstrom
5	A, Ado	adenosine*
6	aa	amino acid (only with numbers)
7	Ab	antibody
8	ABTS	2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
9	Ac	acetyl
10	ac	alternating current
11	ADCC	antibody-dependent cell-mediated cytotoxicity
12	Ade	adenine
13	af	audio frequency
14	Ag	antigen
15	AIDS	acquired immunodeficiency syndrome
16	Ala	alanine
17	AML	acute myeloid leukemia
18	AMP, ADP, ATP, dAMP, ddATP, GTP, etc.	For the respective 5’ phosphates of adenosine and other nucleosides, add 2’-, 3’-, or 5’- when needed for contrast
19	Amt	Amount
20	ANOVA	analysis of variance
21	AP-1	activator protein 1
22	APC	antigen-presenting cell
23	Approx	Approximately
24	Arg	arginine
25	Asn	asparagine
26	Asp	aspartic acid
27	Asx	asparagine or aspartic acid
28	at. wt.	atomic weight
29	ATP	adenosine triphosphate (also ADP, AMP, CMP, CTP, GDP, GMP, GTP, ITP, NTP, TMP, UDP and UTP)
30	ATPase, dGTPase, etc.	Adenosine triphosphatase, deoxyguanosine triophosphatase, etc
31	Avg	Average
32	AZT	3’-azido-3-deoxythymidine
33	B.P.	before present
34	b.p.	boiling point
35	BAL	Bronchoalveolar lavage
36	BALT	bronchus-associated lymphoid tissue
37	BAPTA-AM	1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester
38	bcc	body-centered-cubic
39	BCG	Bacillus Calmette Guerin
40	BCR	B cell receptor
41	BM	Bone marrow
42	bp	base pair (only with numbers)
43	BrdU	5-bromo-2’-deoxyuridine
44	BrUra	bromouracil
45	BSA	bovine serum albumin
46	Bu	butyl
47	Bz	benzoyl
48	C	complement
49	C’	activated complement
50	C region	constant region of Ig
51	C, Cyd	cytidine*
52	C/EBP	CCAAT/enhancer-binding protein
53	Calc., calc	calculated
54	cAMP	cyclic AMP
55	CCL	CC chemokine ligand
56	CCR	CC chemokine receptor
57	CD	circular dichroism
58	CD	cluster of differentiation
59	CD40L	CD40 ligand
60	CDC	complement-dependent cytotoxicity
61	cDNA	complementary DNA
62	CDR	complementarity determining region
63	CFA	complete Freund’s adjuvant
64	CFSE	5- (and 6-)carboxyfluorescein diacetate succinimidyl ester
65	CFU	colony-forming unit
66	cGMP	guanosine 3’ 5’-cyclic monophosphate
67	cGy	centiGray (only with numbers)
68	CHAPS	3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
69	CHO	Chinese hamster ovary
70	Ci	Curie (only with numbers)
71	CIITA	class II transactivator
72	CLIP	class II-associated invariant-chain peptide
73	cm	centimeter (only with numbers)
74	CM-cellulose	O-carboxymethylcellulose
75	CML	chronic myelogenous leukemia
76	CMV	cytomegalovirus
77	CNS	central nervous system
78	CoA	coenzyme A
79	Con A	concanavalin A
80	conc.	concentration
81	Concn	Concentration
82	const	constant
83	COSY	correlated spectroscopy
84	CP	chemically pure
85	CpG	cytosine guanine dinucleotide
86	cpm	counts per minute (only with numbers)
87	CR	Complement receptor
88	CREB	cAMP response element binding protein
89	cRNA	complementary RNA
90	CSF	colony-stimulating factor
91	CTL	cytotoxic T lymphocyte
92	CTLA	cytolytic T lymphocyte-associated Ag
93	CXCL	CXC chemokine ligand
94	CXCR	CXC chemokine receptor
95	Cys	cysteine or half-cystine
96	Cyt	cytosine
97	d	deoxy; distilled (as in dH2O)
98	d	deoxy (carbohydrates and nucleotides)
99	d	day (only with numbers)
100	D region	diversity region of Ig or T cell receptor for Ag
101	Da	dalton (only with numbers)
102	DAPI	4’ 6-diamidino-2-phenylindole
103	dATP	2’-deoxyadenosine triphosphate
104	dc	direct current
105	DC	dendritic cell
106	DEAE	diethylaminoethyl
107	DEAE-cellulose	diethylaminoethylcellulose
108	df	degrees of freedom
109	DHFR	dihydrofolate reductase
110	Diam	Diameter
111	DMEM	Dulbecco’s modified Eagle’s medium
112	DMSO	dimethylsulfoxide
113	DNA	deoxyribonucleic acid
114	DNase	deoxyribonuclease
115	DNP	dinitrophenyl
116	dNTP	2’-deoxynucleoside 5’-triphosphate
117	dpm	disintegrations per minute
118	ds	double-stranded (as dsDNA)
119	dT or dThd	thymidine (2'-deoxyribosylthymine)*
120	DTH	delayed-type hypersensitivity
121	dThd	Thymidine
122	DTT	dithiothreitol
123	dUrd	Deoxyuridine
124	E	erythrocyte
125	E. coli	Escherichia coli
126	E/T	Effector-to-target cell (ratio)
127	E/T ratio	effector to target cell ratio
128	E:T ratio	effector to target ratio
129	EAE	Experimental autoimmune encephalomyelitis
130	EBV	Epstein-Barr virus
131	EC50	50% effective concentration
132	ECG	electrocardiogram
133	ECL	enhanced chemiluminescence
134	ED50	50% effective dose
135	EDTA	ethylenediaminetetraacetic acid
136	EGFP	enhanced green fluorescent protein
137	EGTA	ethylene glycol-bis(b-aminoethyl ester)-N,N,N’,N’-tetraacetic acid
138	ELISA	enzyme-linked immunosorbent assay
139	ELISPOT	enzyme-linked immunospot
140	EM	electron microscopy
141	EMBL	European Molecular Biology Laboratory
142	emf	electromotive force
143	EMSA	electrophoretic mobility shift assay
144	EPR	electron paramagnetic resonance
145	equiv. wt.	equivalent weight
146	ER	endoplasmic reticulum
147	ERK	extracellular signal-regulated kinase
148	ESR	electron spin resonance
149	EST	expressed sequence tag
150	Et	ethyl
151	Etd	ethidium
152	Exp., exp	experiment(al)
153	Expt	Experiment
154	Exptl	Experimental
155	f.p.	freezing point
156	Fab	Ag-binding fragment
157	FACS	fluorescence-activated cell sorter
158	F-actin	filamentous actin
159	FAD	flavin-adenine dinucleotide
160	FAM	6-carboxyfluorescein
161	FBS	fetal bovine serum
162	FBS	Fetal bovine serum
163	Fc	IgG fragment crystallizable
164	Fc-gamma R	Receptor for the Fc-gamma part of the IgG molecule
165	FCM	Flow cytometry
166	FcR	Fc receptors (e.g., FcgRI)
167	FCS	fetal calf serum
168	FISH	fluorescence in situ hybridization
169	FITC	fluorescein isothiocyanate
170	FLICE	Fas-associated death domain-like IL-1b-converting enzyme
171	FLIP	FLICE inhibitory protein
172	fMet	formylmethionine
173	fMLP or FMLP	formyl-methionyl-leucyl-phenylalanine
174	FMN	flavin mononucleotide
175	FPLC	fast protein liquid chromatography
176	Fru	fructose
177	Fuc	fucose
178	Fura 2-AM	fura 2-acetoxymethyl ester
179	Fv	IgG fragment variable
180	g	gram (only with numbers)
181	G, Guo	guanosine*
182	Gal	galactose
183	GalN	galactosamine
184	GALT	gut-associated lymphoid tissue
185	GAPDH or G3PDH	glyceraldehyde-3-phosphate dehydrogenase
186	GC, GLC	gas chromatography, gas/liquid chromatography
187	G-CSF	granulocyte CSF
188	GFP	green fluorescent protein
189	Glc	glucose
190	GlcA	gluconic acid
191	GlcA, GlcUA	glucuronic acid
192	GlcN	glucosamine
193	GlcNAc	N-acetylglucosamine
194	Gln	glutamine
195	Glu	glutamic acid
196	Glx	glutamic acid or glutamine
197	Gly	glycine
198	GM-CSF	granulocyte-macrophage colony stimulating factor
199	gp	glycoprotein (e.g., gp100)
200	GPI	glycosylphosphatidylinositol
201	GST	glutathione S-transferase
202	Gua	guanine
203	GVH	Graft-vs.-host (reaction)
204	h	hour (only with numbers)
205	H chain	heavy chain
206	H&E	hematoxylin and eosin
207	HACA	human anti-chimeric antibodies
208	HAHA	human anti-human antibodies
209	HAMA	human anti-mouse antibodies
210	Hb	hemoglobin
211	HbCO	carbon monoxide hemoglobin
212	HbO2	oxyhemoglobin
213	HBSS	Hanks’ balanced salt solution
214	Hepes or HEPES	N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid
215	hf	high frequency
216	hfs	hyperfine structure
217	HHV	human herpes virus
218	His	histidine
219	HIV	human immunodeficiency virus
220	HLA	human leukocyte antigen
221	HPLC	high-pressure or high-performance liquid chromatography
222	HPV	human papillomavirus
223	HRP	horseradish peroxidase
224	HSP	Heat shock protein
225	HSV	herpes simplex virus
226	Ht	Height
227	HUVEC	human umbilical vein endothelial cells
228	I, Ino	inosine*
229	i.d.	intradermal
230	i.d., o.d.	diameter, inside or outside
231	i.m.	intramuscular
232	i.p.	intraperitoneal
233	i.v.	intravenous
234	IC50	50% inhibition/inhibitory concentration
235	ICAM	intercellular adhesion molecule
236	ICOS	inducible costimulator
237	Id	idiotope, idiotype
238	ID50	50% infective dose or 50% inhibiting dose
239	IDO	indoleamine 2, 3-dioxygenase
240	IFA	incomplete Freund’s adjuvant
241	IFN	interferon (e.g., IFN-g)
242	Ig	immunoglobulin
243	IgG, IgM, etc.	immunoglobulin G, M, etc.
244	IgH	Ig heavy chain
245	IkB	inhibitory NF-kB
246	IL	interleukin (e.g., IL-2)
247	Ile	isoleucine
248	IMDM	Iscove’s modified Dulbecco’s medium
249	IMEM	Iscove’s minimal essential medium
250	IPTG	isopropyl b-d-thiogalactoside
251	IR	infrared
252	ITAM	immunoreceptor tyrosine-based activation motif
253	ITIM	immunoreceptor tyrosine-based inhibitory motif
254	IU	international unit (only with numbers)
255	J region	joining region of Ig or T cell receptor for Ag
256	JAK or Jak	Janus kinase
257	JNK	c-Jun N-terminal kinase
258	Ka	association constant
259	kb	kilobase (only with numbers)
260	kbp	kilobase pair (only with numbers)
261	Kd	distribution coefficient; dissociation constant
262	KD	affinity constant
263	kDa	kilodalton (only with numbers)
264	KIR	Killer cell Ig-like receptor
265	L	ligand
266	L chain	light chain; light
267	LAK	lymphokine-activated killer (cells)
268	LB	Luria-Bertani or Lenox broth
269	LC	Langerhans cell
270	LCMV	Lymphocytic choriomeningitis virus
271	LD50	50% lethal dose
272	Leu	leucine
273	LFA	leukocyte (lymphocyte) function-associated Ag
274	LIF	leukemia inhibitory factor
275	lim	limit
276	LN	Lymph node
277	LPS	lipopolysaccharide
278	LTR	long terminal repeat
279	LU	lytic unit
280	Lys	lysine
281	M	molar (only with numbers)
282	m.p.	melting point
283	m.w.	molecular weight
284	mAb	monoclonal antibody
285	MACS	magnetic-activated cell sorting
286	MALDI	matrix-assisted laser desorption ionization
287	MALDI-TOF	matrix-assisted laser desorption ionization-time of flight
288	MALT	mucosa-associated lymphoid tissue
289	Man	mannose
290	MAPK	mitogen-activated protein kinase
291	Mb	myoglobin
292	MbCO	carbon monoxide myoglobin
293	MCP-1	monocyte chemoattractant protein-1
294	M-CSF	macrophage CSF
295	Me	methyl
296	MEK	mitogen-activated protein kinase kinase
297	MEM	minimum essential medium
298	MES	2-(N-morpholino)ethanesulfonic acid
299	Met	methionine
300	mg	milligram (only with numbers)
301	MHC	major histocompatibility complex
302	MIC	Minimal inhibitory concentration
303	min	minute (only with numbers)
304	MIP	macrophage-inflammatory protein
305	ml	milliliter (only with numbers)
306	MLC	mixed lymphocyte culture
307	MLR	mixed leukocyte reaction
308	mM	millimolar (only with numbers)
309	mo	month(s) (only with numbers)
310	mol	mole
311	Mol wt	Molecular weight
312	MOPS	4-morpholinepropanesulfonic acid
313	Mr	relative molecular mass
314	MRI	magnetic resonance imaging
315	mRNA	messenger RNA
316	MS	mass spectrometry or spectroscopy
317	mtDNA	mitochondrial DNA
318	MTT	3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
319	MU	Macrophage
320	MyD88	myeloid differentiating factor 88
321	n	number in study or group
322	N, Nuc	nucleoside (unknown)*
323	NAD	nicotinamide adenine dinucleotide
324	NAD+	Nicotinamide adenine dinucleotide, oxidized
325	NADH	Nicotinamide adenine dinucleotide, reduced
326	NaDodSO4	sodium dodecyl sulfate
327	NADP	NAD phosphate
328	NADP+, NADPH	nicotinamide-adenine dinucleotide phosphate
329	NADPH	Nicotinamide adenine dinucleotide phosphate, reduced
330	NADPH+	Nicotinamide adenine dinucleotide phosphate, oxidized
331	NBT	nitroblue tetrazolium
332	NCI	National Cancer Institute
333	ND	not determined
334	NDP	nucleoside 5’-diphosphate
335	NF	nuclear factor
336	NFAT or NF-AT	nuclear factor of activated T cells
337	NF-kB	nuclear factor kB
338	NIH	National Institutes of Health
339	Ni-NTA	nickel-nitrilotriacetic acid
340	NK	natural killer (cells)
341	NKT	Natural killer T (cell)
342	NMN	nicotinamide mononucleotide
343	NMP	nucleoside 5’-monophosphate
344	NMR	nuclear magnetic resonance
345	NO	nitric oxide
346	No.	Number
347	NOD	nonobese diabetic
348	NOESY	nuclear Overhauser effect/enhancement spectroscopy
349	NP40	Nonidet-P40
350	NS	not significant
351	nt	nucleotide (only with numbers)
352	nt	nucleotide (only with numbers)
353	OAc	acetate
354	Obs., obs	observed
355	OCT	octamer-binding factor
356	OD	optical density
357	oligo(dT), etc.	Oligodeoxythymidylic acid, etc.
358	ORD	optical rotatory dispersion
359	ORF	open reading frame
360	OVA	ovalbumin
361	p	probability
362	P	probability
363	P or p	phosphate (in compounds)
364	PAGE	polyacrylamide gel electrophoresis
365	PBL	peripheral blood lymphocyte
366	PBMC	peripheral blood mononuclear cell
367	PBS	phosphate-buffered saline
368	PCR	polymerase chain reaction
369	PCR-RFLP	PCR-restriction fragment length polymorphism
370	PE	phycoerythrin
371	PECAM-1	platelet endothelial cell adhesion molecule-1
372	PEG	polyethylene glycol
373	PEI-cellulose	polyethylenimine-cellulose
374	PerCP	peridinin chlorophyll protein
375	PFC	Plaque-forming cell
376	PFU or pfu	plaque-forming unit
377	PG	prostaglandin
378	Ph	phenyl
379	PHA	phytohemagglutinin
380	Phe	phenylalanine
381	pI	isoelectric point
382	Pi	phosphate (inorganic)
383	pI	isoelectric point
384	PI	propidium iodide
385	PI3K	phosphatidylinositol 3-kinase
386	PIPES	piperazine-N
387	Pipes	1,4-piperazinediethanesulfonic acid
388	PK, PKC	protein kinase, protein kinase C
389	PKC	Protein kinase C
390	PMA	phorbol myristate acetate
391	pmf	proton-motive force
392	PMN	Polymorphonuclear (cell, leukocyte)
393	PMSF	phenylmethylsulfonyl fluoride or phenylmethanesulfonyl fluoride
394	poly(A), poly(dT), etc.	Polyadenylic acid, polydeoxythymidylic acid, etc.
395	PPD	Purified protein derivative of tuberculin
396	Pr	propyl
397	Prepn	Preparation
398	Pro	proline
399	PWM	pokeweed mitogen
400	QED	quantum electrodynamics
401	r	recombinant, (e.g., rIFN-g)
402	R	receptor (e.g., IL-2R)
403	RACE	rapid amplification of cDNA end
404	RAG	recombination-activating gene
405	RANTES	regulated upon activation
406	RBC	red blood cell
407	rDNA, rRNA	ribosomal DNA, ribosomal RNA
408	resp (mathematics)	respectively
409	rf	radio frequency
410	RFLP	restriction fragment length polymorphism
411	RIA	radioimmunoassay
412	Rib	ribose
413	rms	root-mean-square
414	RNA	ribonucleic acid
415	RNase	ribonuclease
416	rpm	revolutions per minute
417	RPMI	Roswell Park Memorial Institute (culture media)
418	rRNA	ribosomal RNA
419	RT-PCR	reverse transcriptase polymerase chain reaction
420	s	second (only with numbers)
421	s.c.	subcutaneous(ly)
422	SCID	severe combined immunodeficiency
423	SD	standard deviation
424	SDS	sodium dodecyl sulfate
425	SDS-PAGE	sodium dodecyl sulfate polyacrylamide gel electrophoresis
426	SE	standard error
427	SEM	standard error of the mean
428	Ser	serine
429	SHIP	src homology 2-containing inositol 5’ phosphatase
430	sIg	Surface immunoglobulin
431	SIV	simian immunodeficiency virus
432	SLE	Systemic lupus erythematosus
433	SN	Supernatant
434	Sp gr	Specific gravity
435	sp. act.	specific activity
436	SPF	Specific pathogen free
437	SRBC	sheep red blood cells
438	ss	single-stranded (e.g., ssDNA)
439	SSC	standard saline citrate
440	STAT	signal transducer and activator of transcription
441	SV40	simian virus 40
442	T or Thd	ribosylthymine
443	t1/2	half-life, half-time
444	TAMRA	5-(and 6)-carboxytetramethylrhodamine
445	TAP	transporter associated with Ag processing
446	Tat	terminal deoxynucleotidyltransferase
447	TBS	Tris-buffered saline
448	TBST	TBS with Tween 20
449	TCA	trichloroacetic acid
450	TCR	T cell receptor
451	TdR	thymidine deoxyribose (also UdR, AdR)
452	TdT	terminal deoxynucleotidyltransferase
453	Temp	Temperature
454	TFA	trifluoroacetic acid
455	Tg	Transgenic
456	TGF	transforming growth factor
457	Th	T helper (cell)
458	Thr	threonine
459	Thy	thymine
460	TIL	tumor-infiltrating lymphocyte
461	TLC	thin-layer chromatography
462	TLR	Toll-like receptor
463	TNF	tumor necrosis factor
464	TNP	trinitrophenyl
465	Tr	Trace
466	TRAIL	TNF-related apoptosis-inducing ligand
467	Tris	tris(hydroxymethyl)aminomethane
468	tRNA	transfer RNA
469	Trp	tryptophan
470	TUNEL	Tdt-mediated dUTP nick end labeling
471	Tyr	tyrosine
472	U	unit (only with numbers)
473	U, Urd	uridine*
474	UHF	ultrahigh frequency
475	Ura	uracil
476	UTR	untranslated region
477	UV	ultraviolet
478	V region	variable region of Ig
479	V(D)J	variable diversity joining
480	v/v	volume to volume ratio (%)
481	Val	valine
482	VCAM	vascular cell adhesion molecule
483	VLA	very late activation Ag
484	vol	volume
485	Vs	Versus
486	VSV	vesicular stomatitis virus
487	W	watt (only with numbers)
488	WBC	white blood cell
489	wk	week (only with numbers)
490	WT	wild type
491	Wt	Weight
492	Xaa	unknown or "other" amino acid
493	xid	X-linked immunodeficiency
494	yr	year (only with numbers)
495	ZAP70	ζ-associated protein 70 (or ζ-chain-associated protein 70)
496	μg	microgram (only with numbers)
497	μl	microliter (only with numbers)